WO2011146806A1

WO2011146806A1 - Methods for reducing anesthetic-inducible epileptogenic and neurotoxic effects

Info

Publication number: WO2011146806A1
Application number: PCT/US2011/037289
Authority: WO
Inventors: Anatoly Eugeny Martynyuk; Christoph Nikolaus Seubert; Nikolaus Gravenstein
Original assignee: University Of Florida Research Foundation, Inc.
Priority date: 2010-05-21
Filing date: 2011-05-20
Publication date: 2011-11-24

Abstract

The disclosure encompasses methods of reducing a neural activity in an animal or human subject. The neural activity may be a seizure that may be, but is not necessarily, an inducible side-effect of an anesthetic in an animal or human subject. The methods include administering to an animal or human subject an anesthetic together with a therapeutic agent selected from an inhibitor of the Na⁺-K⁺-2Cl^- symport ion co-transporter 1 (NKCC1)agent, a mineralcorticoid receptor (MR) antagonist, or an agonist of an oxytocin receptor or a derivative thereof. The therapeutic agent can be be administered before or with an anesthetic, thereby reducing the likelihood of the onset of a non-anesthesizing effect of the anesthetic or the severity thereof. A suitable mineralcorticoid receptor (MR) antagonist can be 17-spironolactone, and the oxytocin receptor agonist can be, but not limited to, oxytocin, carbetocin, and the like. The neural activity reduced by the methods of the disclosure can include the onset or level of a seizure, a neurotoxicity, a behavioral effect, a cognitive effect.

Description

METHODS FOR REDUCING ANESTHETIC-INDUCIBLE EPILEPTOGENIC AND

NEUROTOXIC EFFECTS

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims priority to U.S. Provisional Patent Application Serial No.:

61 /346,990, entitled "BUMETANIDE ALLEVIATES EPILEPTOGENIC AND NEUROTOXIC EFFECTS OF SEVOFLURANE IN NEONATAL RAT BRAIN" filed on May 21 , 2010, and to U .S. Provisional Patent Application Serial No.: 61/450, 164, entitled "BUMETANIDE

ALLEVIATES EPILEPTOGENIC AND NEUROTOXIC EFFECTS OF SEVOFLURANE IN NEONATAL RAT BRAIN" filed on March 8, 201 1 the entireties of which are hereby

incorporated by reference.

TECHNICAL FIELD

The present disclosure is generally related to methods of reducing or preventing at least one adverse neural activity including side-effects of the application to a subject animal or human of an anesthetic.

BACKGROUND

Each year, millions of term and pre-term neonates and fetuses of pregnant mothers are exposed to general anesthetics during medical procedures that require anesthesia.

Retrospective epidemiological analyses (DiMaggio et ai , (2009) J. Neurosurg. Anesthesiol. 21 : 286-29 ; Wilder et ai , (2009) Anesthesiology 1 10: 796-804), and animal studies (Jevtovic-

Todorovic et at., (2003) J. Neurosci. 23: 876-882; Zhang ef ai , (2008) Neurosci. Lett. 447:109- 1 14; Satomoto et ai , (2009) Anesthesiology 1 10: 628-637; Stratmann er a/. , (2009)

Anesthesiology. 1 10: 849-861 ; Loepke ef a/. , (2009) Anesth. Analg. 108: 90-104; Brambrink et ai , (2010) Anesthesiology 112: 834-841 ; Shu et ai , (2010) Anesthesiology 13: 360-368;

Edwards ef ai. , (2010) Anesthesiology 1 12: 567-575) indicate that exposure to general anesthesia during the early period of brain development may result in immediate and delayed brain abnormalities, such as epileptic seizures, cytotoxicity, and impaired cognition.

The mechanisms mediating the adverse actions of general anesthetics, and even more so conditions that may specifically affect the severity of the anesthetics' side effects are essentially unknown. The predominant hypothesis that has been explored in almost all published animal studies thus far is that anesthetic-induced inhibition of neuronal activity in the early stages of brain development results in neuronal death, altered neurogenesis, and impaired cognitive function. This hypothesis, however, is in disagreement with a number of experimental findings. For instance, oxytocin that is released in increased amounts during the short period of birth inhibits neuronal activity and exerts, not toxic, but neuroprotective effects on the fetal brain.

Hemodynamic stability, lack of respiratory irritation and, short-lasting action make sevoflurane one of the most widely used general anesthetics, particularly in pediatric anesthesia. Unfortunately, sevoflurane, like some other general anesthetics, has been reported to cause epileptiform electroencephalographic activity and seizure-like movements (Zeiler & Kaplan (2008) Seizure 17: 665-667; Welborn et al. , (1996) Anesth. Analg. 83: 917-920;

Veyckemans F: (2001 ) Curr. Opin. Anaesthesiol. 14: 339-343; Constant et al., Paediatr.

Anaesth. (2005) 15: 266-274; Mohanram et al., (2007) Can. J. Anaesth. 54: 657-661 ; Griffeth & Mehra (2007) J. ECT. 23: 177-178; Harrison JL: (1986) Analg. 65: 1235-1236; McManus KF: (1992) Anaesth. Intensive Care 20: 245). The hyperexcitatory events in neonates and infants are of tremendous concern as they may potentially result in delayed neurological and cognitive defects (Tekgul et al., (2006) Pediatrics 1 17: 1270-1280). Recent data indicate that a single episode of neonatal seizures is sufficient to cause permanent alterations in memory long after the initial seizure episode in a neonatal rat model (Cornejo et al. , (2007) Ann. Neurol. 61 : 41 1 - 26).

The major known effects of sevoflurane in the brain include activation of various two- pore domain K⁺ channels, depression of glutamate release, inhibition of nicotinic acetylcholine receptors and enhancement of many types of gamma-aminobutyric acid (GABA)_A and strychnine-sensitive glycine receptors (Ishizeki ef al., (2008) Anesthesiology 108: 447-456; Solt & Forman (2007) Curr. Opin. Anaesthesiol. 20: 300-306; Alkire et al., (2007) Anesthesiology 107: 264-272; Putzke et al., (2007) Am. J. Physiol. Cell Physiol. 293: C1319-1326). All these effects, at least in their classical understanding, lead to inhibition and induction of the anesthetic state, but by themselves should not cause excessive excitatory phenomena as may be observed during and immediately after anesthesia with sevoflurane. On the other hand, the changes in the transmembrane gradient of CI^", which is the charge carrier through GABA_A and strychnine-sensitive glycine receptor channels (Staley ef al., ( 995) Science 269: 977-981 ; Bormann ef al., (1987) J. Physiol. 385: 243-286) can reverse the output of activation of these receptors from inhibitory to excitatory and, therefore, may play an important role in the hyperexcitation phenomena associated with sevoflurane anesthesia. The observations that all general anesthetics that have a GABA-ergic component of action may cause episodes of hyperexicitation indirectly support this possibility(Zeiler & Kaplan (2008) Seizure 17: 665-667; Welborn et al., (1996) Anesth. Analg. 83: 917-920; Veyckemans F: (2001 ) Curr. Opin.

Anaesthesiol. 14: 339-343; Constant ef al. , Paediatr. Anaesth. (2005) 15: 266-274; Mohanram ef al., (2007) Can. J. Anaesth. 54: 657-661 ; Griffeth & Mehra (2007) J. ECT. 23: 177-178; Harrison JL: (1986) Analg. 65: 1235-1236; McManus KF: (1992) Anaesth. Intensive Care 20: 245).

In comparison with mature neurons, neurons at early stages of postnatal development have significantly elevated intracellular concentrations of CI^", [Cl^"]i, and as a result the equilibrium potential for CI^", E_Ci, is more positive than the resting membrane potential. Therefore, during early development, activation of GABA_A receptors results in CI^" efflux and neuronal depolarization (Owens ef al. , (1996) J. Neurosci. 16: 6414-6423; Zhang ef al., (2006) J. Neurophysiol. 95: 2404-2416; Dzhala er a/., (2005) Nat. Med. 1 1 : 1205-1213). In fact, GABA_A receptor activation is recognized as a major source of excitation at early stages of brain development (Ben-Ari ei a/., (2007) Physiol. Rev. 87:1215-1284).

Uptake of CI^" by the Na⁺-K⁺-2CI^" cotransporter isoform 1 (NKCC1 ) has been shown to provide the driving force for depolarizing GABA_A receptor-mediated responses in immature neurons (Bormann et a/., (1987) J. Physiol. 385: 243-286; Owens ef a/., (1996) J. Neurosci. 16: 6414-6423; Zhang ef a/., (2006) J. Neurophysiol. 95: 2404-2416; Dzhala ef a/., (2005) Nat. Med. 1 1 : 1205-1213). The peak expression of NKCC1 in rodents is around postnatal days 5-7.

Consistent with the specific role of NKCC1 in the depolarizing effects of GABA, GABA-mediated depolarization in immature neurons was shown to be blocked by bumetanide, a specific inhibitor of NKCC1 at low doses (Yamada ef a/., (2004) J. Physiol. 557(Pt 3): 829-841 ). Bumetanide also inhibits cortical seizure activity in neonatal rats in vitro and in vivo. The ontogenetic shift to a hyperpolarizing action of GABA is caused by a concomitant developmental down-regulation of NKCC1 and an up-regulation of the K⁺-CI^" co-transporter isoform 2 (KCC2) (Rivera ef a/., (1999) Nature 397: 251-255; Lee ef a/., (2005) Eur. J. Neurosci. 21 : 2593-2599). KCC2 expression is not detectable in rat neurons after birth, and its expression during the second postnatal week is associated with a progressive negative shift in the reversal potential of GABA_A receptor- mediated responses and a switch in the action of GABA from excitatory to inhibitory (Yamada ef a/., (2004) J. Physiol. 557(Pt 3): 829-841 ; Rivera ef a/., (2005) J. Physiol. 562: 27-36).

SUMMARY

Briefly described, embodiments of this disclosure, among others, encompass methods of inhibiting the onset or the severity of seizures, neurotoxicity, cognitive

impairment, and the like and which can be, but not necessarily, inducible side-effects of anesthesia. Embodiments of the methods of the disclosure encompass the administration of a Na⁺-K⁺-2C co-transporter isoform 1 (NKCC1 ) inhibitor simultaneously with the anesthetic, or before anesthesia. In particular, the methods of the disclosure encompass the use of the Na⁺-K⁺-2CI^" co-transporter isoform 1 (NKCC1 ) inhibitor butemanide, although the use of other agents having the anti-NKCC1 activity is within the scope of the disclosure. The disclosure further encompasses the administration of an aldosterone receptor antagonist or an oxytocin receptor agonist that may also reduce the severity of such as a seizure induced by an anesthetic, or due to a non-anesthetic cause.

One aspect of the present disclosure, therefore, encompasses methods of reducing a non-anesthesizing side-effect of an anesthetic in an animal or human subject, the method comprising: administering to an animal or human subject an effective dose of anesthetic; and administering to the animal or human subject at least one therapeutic agent selected from the group consisting of: a therapeutic agent characterized as decreasing the intracellular amount of CI^" in a recipient subject animal or human; a mineralcorticoid receptor (MR) antagonist, and an oxytocin receptor agonist; wherein the therapeutic agent can be administered before or with the anesthetic, thereby reducing the level of a non-anesthesizing side-effect of the anesthetic in the animal or human subject.

In embodiments of this aspect of the disclosure, the at least one therapeutic agent characterized as decreasing the intracellular amount of CI^" in the recipient subject animal or human is an inhibitor of the Na⁺-K⁺-2CI^" symport ion co-transporter 1 (NKCC1 ).

In embodiments of this aspect of the disclosure, the inhibitor of the Na⁺-K⁺-2CI^" symport ion co-transporter 1 (NKCC1 ) is selected from the group consisting of: furosemide, butmetanide (3-butylamino-4-phenoxy-5-sulfamoyl-benzoic acid), ethacrynic acid, and benzmetanide, tripamide, or derivatives thereof.

In some embodiments of this aspect of the disclosure, the inhibitor of the Na⁺-K⁺-2CI^" symport ion co-transporter 1 (NKCC1 ) is butmetanide.

In embodiments of this aspect of the disclosure, the inhibitor of the Na⁺-K⁺-2CI^" symport ion co-transporter 1 (NKCC1 ) can be butmetanide and the anesthetic is

sevoflurane.

In embodiments of this aspect of the disclosure, the mineralcorticoid receptor (MR) antagonist can be an aldosterone antagonist.

In embodiments of this aspect of the disclosure, the mineralcorticoid receptor (MR) antagonist can be spironolactone or a derivative thereof. In some embodiments of this aspect of the disclosure, the mineralcorticoid receptor (MR) antagonist is 17-spironolactone.

In embodiments of this aspect of the disclosure, the oxytocin receptor agonist can be selected from the group consisting of: oxytocin, carbetocin, or an analog or a derivative thereof.

In embodiments of this aspect of the disclosure, the anesthetic increases

GABA_A/glycine receptor activity in the recipient subject animal or human.

In embodiments of this aspect of the disclosure, the anesthetic is isoflurane or sevoflurane.

In embodiments of this aspect of the disclosure, the side-effect of the anesthetic can be selected from the group consisting of: an induction of a seizure, a neurotoxicity, a behavioral effect, a cognitive effect, or any combination thereof.

Another aspect of the disclosure encompasses embodiments of a method of reducing a neural activity in an animal or human subject, the method comprising:

administering to an animal or human subject an effective dose of a mineralcorticoid receptor (MR) antagonist or an oxytocin receptor agonist; wherein the mineralcorticoid receptor (MR) antagonist or oxytocin receptor agonist reduces the level of a neural activity in the animal or human subject.

In embodiments of this aspect of the disclosure, the neural activity is a seizure. In some embodiments of this aspect of the disclosure, the seizure is not a side-effect of an anesthetic.

In embodiments of this aspect of the disclosure, the mineralcorticoid receptor (MR) antagonist can be a spironolactone, or a derivative thereof.

In embodiments of this aspect of the disclosure, the oxytocin receptor agonist can be selected from the group consisting of: oxytocin, carbetocin , or an analog or a derivative thereof.

Yet another aspect of the disclosure encompasses embodiments of a method of determining the likelihood of an animal or human subject developing a side-effect of an anesthetic administered to said subject, the method comprising determining whether the subject has an abnormality in a level of aldosterone or oxytocin compared to a normal level, or an abnormality in the physiological function or response thereto of aldosterone or oxytocin in the animal or human subject, wherein the presence of the abnormality indicates the likelihood of an animal or human subject developing a side-effect of an anesthetic

administered to said subject.

BRIEF DESCRIPTION OF THE DRAWINGS

Further aspects of the present disclosure will be more readily appreciated upon review of the detailed description of its various embodiments, described below, when taken in conjunction with the accompanying drawings.

Fig. 1 A is a digital image of EEG electrode placement for continuous EEG recordings from bilateral occipital (EEG 1 ) and right frontal (EEG2) regions using an EEG/electromyogram system.

Fig. 1 B illustrates examples of EEG recordings from postnatal days 4 (P4) and 17 (P17) rats during the start of anesthesia induction with 6% sevoflurane and during anesthesia maintenance with 2.1 % sevoflurane.

Fig. 1 C illustrates EEG traces showing seizures, recorded from a 5-day-old (P5) male rat during anesthesia with 2.1 % of sevoflurane.

Fig. 2A illustrates the experimental protocol to investigate whether the Na⁺-K⁺-2CI~ co- transporter 1 (NKCC 1 ) inhibitor bumetanide depresses seizures during sevoflurane anesthesia. Two groups of animals (P4-P9) received either bumetanide (5 pmol/kg, intraperitoneally), or an equal volume of saline, 15 min prior to anesthesia with 6% and 2.1 % sevoflurane that lasted for 3 min and 60 min, respectively.

Fig. 2B illustrates examples of electroencephalogram recordings during 2.1 %

sevoflurane anesthesia in a P4 rat.

Fig. 2C shows a series of graphs summarizing the data of the experiments of Figs. 2A and 2B. Bumetanide decreased total time of seizure-like activity and number of episodes of seizures. *, P<0.05 vs. saline plus sevoflurane. Figs. 3A and 3B show a series of electroencephalographic (EEG) recordings of seizures during emergence from 3 hour-long anesthesia with 2.1 % sevoflurane.

Fig. 3A shows EEG recordings from a 14-day-old male rat. Dotted lines indicate the episode of the EEG that corresponds to the fragments of EEG recording shown above.

Fig. 3B illustrates power spectra for each stage of the experiment shown in Fig. 3A.

Fig. 4A illustrates the experimental protocol for showing isoflurane anesthesia of neonatal rats increases caspase-3 activation, and which is diminished by pretreatment with bumetanide. Postnatal day 4 (P4) rats were pre-treated either with 5 μΓηοΙ/kg bumetanide or an equal volume of saline. Rats in the control group did not undergo anesthesia on P4 before exposure to 6 hours of isoflurane (1 .2%) anesthesia. Brains were isolated on P 5 for cleaved caspase-3 evaluation.

Fig. 4B is a digital image of a representative Western blot analysis of cleaved caspase-3 in the brains of all three groups of rats.

Fig. 4C shows a histogram illustrating the densitometric analysis of Western blot images of cleaved caspase-3. Densities of γ-tubulin blots from the same tissue sample were taken as 100%. *, PO.05 vs. control.

Fig. 5A illustrates the experimental protocol for showing that sevoflurane anesthesia of neonatal rats increases caspase-3 activation, which is diminished by pretreatment with bumetanide. Postnatal day 4 (P4) rats were pre-treated either with 5 μΓηοΙ/kg bumetanide (sevoflurane + bumetanide; n=4) or an equal volume of saline (sevoflurane; n=4) before exposure to 6 hours of sevoflurane (2.1 %) anesthesia. Rats in the control group (control; n=5) did not undergo anesthesia on P4. Brains were isolated on P 5 for cleaved caspase-3 evaluation.

Fig. 5B is a digital image of a representative Western blot analysis of cleaved caspase-3 in the brain of all three groups of rats to illustrate band intensities.

Fig. 5C shows a histogram showing the densitometric analysis of the Western blot images of cleaved caspase-3 shown in Fig. 5B. Densities of γ-tubulin blots from the same tissue sample were taken as 100%. *and ^#, P<0.05 vs. control and sevoflurane plus bumetanide, respectively.

Fig. 6A is a series of digital images illustrating an immunostaining analysis of sevoflurane anesthesia of neonatal rats increasing caspase-3 activation, which is diminished by pretreatment with bumetanide. Immunofluorescent staining images (DAPI, all cells; NeuN, neurons; activated caspase-3, all cells).

Fig. 6B shows a pair of histograms counting of activated caspase-3 co-localized with neurons (NeuN) in the cortex of P5 rats treated at P4. Immunostaining was performed in 20 μΜ thick slices from the cortex with the same anatomical location for all animals. Counting was performed in 1 mm² area. *, P<0.05 vs. control for B and C.

Fig. 7 A illustrates the experimental protocol involving an assessment of Prepulse Inhibition (PPI) of acoustic startle response (sensorimotor gating function), which is deficient in many neuropsychiatric disorders. The SR-LAB apparatus and accompanying software (San Diego Instruments, San Diego, CA) were used to perform the tests. The PPI of startle was disrupted in juvenile rats that were exposed to isoflurane at P4. The PPI of startle was measured at P21-P24 and P31-35. Bumetanide (5 pmol/kg, I. P.) administered prior to isoflurane, decreased these effects of sevoflurane. Rats in the control group did not undergo anesthesia on P4.

Fig. 7B shows a pair of histograms showing %PPI in different treatment groups. ^*, P<0.05 vs Control.

Figs. 8A-8C illustrate that sevoflurane anesthesia at postnatal days 4-5 (P4-P5) causes abnormalities in prepulse inhibition (PPI) of acoustic startle response and grooming behavior in rats measured at P21 -P25. These abnormalities were diminished by bumetanide, administered prior to sevoflurane anesthesia.

Fig. 8A illustrates the experimental protocol. Rats in the control groups did not undergo anesthesia on P4-P5.

Fig. 8B shows a histogram showing %PPI of startle in different treatment groups: control (n=1 1 ), saline + sevoflurane (n=14) and bumetanide + sevoflurane (n=15). ^*, P<0.05 vs Control. PP5-PP15 - prepulse intensities in dB above background.

Fig. 8C shows a histogram showing time spent grooming by three treatment groups: 1 ) control (n=17); saline + sevoflurane (n=12) and bumetanide + sevoflurane (n=7). *, P<0.05 vs Control.

Figs 9A-9C illustrate that the mineralocorticoid receptor antagonist, spironolactone, does not prevent cortical seizures in P4-P6 rats during sevoflurane anesthesia.

Fig. 9A illustrates the experimental protocol.

Fig. 9B illustrates an example of EEG recording of cortical seizures in P5 rat during 2.1 % sevoflurane anesthesia after the administration of spironolactone.

Fig. 9C shows a series of histograms showing properties of cortical seizures during 2.1 % sevoflurane anesthesia after administration of spironolactone (n=6).

Figs 10A-10C illustrate that spironolactone administered prior to sevoflurane anesthesia depresses activation of caspase-3 in rat cerebral cortex.

Fig. 10A illustrates the experimental protocol.

Fig. 10B shows digital images of a Western blot analysis of cleaved caspase-3 and γ- tubulin in the cerebral cortex tissue of P4 rats. Fig. 10C shows a histogram of a densitometric analysis of cleaved caspase-3 in the cortex tissue from four treatment groups. Rats in the two treatment groups were exposed to sevoflurane anesthesia either in the absence (DMSO + sevoflurane; n=5) or presence of spironolactone (aldosterone + sevoflurane; n=8). Rats in the control groups (DMSO; n=4) and (spironolactone; n=3) did not undergo anesthesia on postnatal day 4. Densities of γ-tubulin blots from the same tissue sample were taken as 100%. *, P<0.05.

Figs. 1 A-1 1 C illustrate that the pretreatment of rats with spironolactone prior to sevoflurane anesthesia diminishes impairment of prepulse inhibition (PPI) of startle but not grooming behavior.

Fig. 1 1 A illustrates the experimental protocol.

Fig. 1 1 B and 1 1 C show histograms showing %PPI and time spent grooming, respectively, in four treatment groups: Rats in the two treatment groups were exposed to sevoflurane anesthesia either in the absence (DMSO + sevoflurane; n=6) or presence of spironolactone (spironolactone + sevoflurane; n=6). Rats in the control group (spironolactone; n=6) did not undergo anesthesia at P4. PP5-PP15 - prepulse intensities in dB above background. *, P<0.05.

Figs. 12A-12D illustrate that exogenous aldosterone exacerbates cortical seizures in P4- P6 rats, but does not affect EEG activity in P17-P21 rats during anesthesia with sevoflurane.

Fig. 12A illustrates the experimental protocol.

Fig. 12B illustrates an example of EEG recording of cortical seizures in a P6 rat during

2.1 % sevoflurane anesthesia after administration of aldosterone. A fragment of the same EEG marked by the horizontal bar is shown below at an expanded time scale.

Fig. 12C shows a series of histograms showing properties of cortical seizures during 2.1 % sevoflurane anesthesia before and after the administration of aldosterone in the same rats (n=4). *, P<0.05 vs. 60 min period prior to aldosterone administration.

Fig. 12D shows an example of EEG recordings from the P1 9 rat during 2.1 % sevoflurane anesthesia before (1 ) and after (2) administration of aldosterone.

Figs. 13A- 3C illustrate that exogenous aldosterone increases activation of caspase-3 in the cerebral cortex of P4 rats anesthetized with sevoflurane.

Fig. 13A illustrates the experimental protocol.

Fig. 1 3B shows a digital image of a Western blot analysis of cleaved caspase-3 and γ- tubulin in the brain cortex of P4 rats.

Fig. 13C shows a histogram of the densitometric analysis of cleaved caspase-3 in the cortex tissue from four treatment groups. Rats in the two treatment groups were exposed to sevoflurane anesthesia either in the absence (DMSO + sevoflurane; n= 1 1 ) or presence of aldosterone (aldosterone + sevoflurane; n=1 1 ). Rats in the control groups (DMSO; n=4) and (aldosterone; n=6) did not undergo anesthesia on postnatal day 4. Densities of γ-tubulin blots from the same tissue sample were taken as 100%. *, P<0.05.

Figs. 14A-14C illustrate that exogenous aldosterone administered to P4 rats prior to sevoflurane anesthesia exacerbates the impairment of prepulse inhibition (PPI) of startle but not grooming behavior measured at P24.

Fig. 14A illustrates the experimental protocol.

Fig. 14B shows a histogram showing %PPI in four treatment groups: Rats in two treatment groups were exposed to sevoflurane anesthesia either without (DMSO + sevoflurane; n=15) or with administered aldosterone (aldosterone + sevoflurane; n=15). Rats in the control groups (DMSO; n=10, and aldosterone; n=10) did not undergo anesthesia at P4. PP5-PP15 - prepulse intensities in dB above background. *, P<0.05.

Fig. 14C shows a histogram showing the time spent grooming in four treatment groups: Rats in two treatment groups were exposed to sevoflurane anesthesia either without (DMSO + sevoflurane; n=15) or with administered aldosterone (aldosterone + sevoflurane; n=15). Rats in the control groups (DMSO; n=10, and aldosterone; n=10) did not undergo anesthesia at P4. PP5-PP15 - prepulse intensities in dB above background. *, P<0.05.

Figs. 15A and 15B illustrate that oxytocin and carbetocin depress cortical seizures in P4- P6 rats during anesthesia with sevoflurane.

Fig. 15A illustrates the experimental protocol.

Fig. 15B shows a series of histograms showing properties of cortical seizures during

2.1 % sevoflurane anesthesia in rats from three treatment groups: oxytocin (n=10); carbetocin (n=6) and saline (n=4). *, P<0.05 versus oxytocin and carbetocin.

Figs. 16A-16C illustrate that carbetocin administered to P4 rats prior to sevoflurane anesthesia diminishes impairment of prepulse inhibition of startle and decreases time spent grooming in rats measured at P24.

Fig. 16 illustrates the experimental protocol.

Fig. 16B shows a histogram showing %PPI in two treatment groups: Saline (n=3), and Carbetocin (n=6). *, P<0.05 vs saline. PP5-PP15 - prepulse intensities in dB above background.

Fig. 16C shows a histogram showing time spent grooming by the same rats as in B. *, P<0.05 versus saline.

The drawings are described in greater detail in the description and examples below. The details of some exemplary embodiments of the methods and systems of the present disclosure are set forth in the description below. Other features, objects, and advantages of the disclosure will be apparent to one of skill in the art upon examination of the following description, drawings, examples and claims. It is intended that all such

additional systems, methods, features, and advantages be included within this description, be within the scope of the present disclosure, and be protected by the accompanying claims. DETAILED DESCRIPTION

Before the present disclosure is described in greater detail, it is to be understood that this disclosure is not limited to particular embodiments described , and as such may, of course, vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to be limiting, since the scope of the present disclosure will be limited only by the appended claims.

Where a range of values is provided, it is understood that each intervening value, to the tenth of the unit of the lower limit unless the context clearly dictates otherwise, between the upper and lower limit of that range and any other stated or intervening value in that stated range, is encompassed within the disclosure. The upper and lower limits of these smaller ranges may independently be included in the smaller ranges and are also encompassed within the disclosure, subject to any specifically excluded limit in the stated range. Where the stated range includes one or both of the limits, ranges excluding either or both of those included limits are also included in the disclosure.

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs. Although any methods and materials similar or equivalent to those described herein can also be used in the practice or testing of the present disclosure, the preferred methods and materials are now described.

All publications and patents cited in this specification are herein incorporated by reference as if each individual publication or patent were specifically and individually indicated to be incorporated by reference and are incorporated herein by reference to disclose and describe the methods and/or materials in connection with which the publications are cited. The citation of any publication is for its disclosure prior to the filing date and should not be construed as an admission that the present disclosure is not entitled to antedate such publication by virtue of prior disclosure. Further, the dates of publication provided could be different from the actual publication dates that may need to be independently confirmed.

As will be apparent to those of skill in the art upon reading this disclosure, each of the individual embodiments described and illustrated herein has discrete components and features which may be readily separated from or combined with the features of any of the other several embodiments without departing from the scope or spirit of the present disclosure. Any recited method can be carried out in the order of events recited or in any other order that is logically possible.

Embodiments of the present disclosure will employ, unless otherwise indicated, techniques of medicine, organic chemistry, biochemistry, molecular biology, pharmacology, and the like, which are within the skill of the art. Such techniques are explained fully in the literature.

It must be noted that, as used in the specification and the appended claims, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to "a support" includes a plurality of supports. In this specification and in the claims that follow, reference will be made to a number of terms that shall be defined to have the following meanings unless a contrary intention is apparent.

As used herein, the following terms have the meanings ascribed to them unless specified otherwise. In this disclosure, "comprises," "comprising," "containing" and "having" and the like can have the meaning ascribed to them in U.S. Patent law and can mean " includes," "including," and the like; "consisting essentially of or "consists essentially" or the like, when applied to methods and compositions encompassed by the present disclosure refers to compositions like those disclosed herein, but which may contain additional structural groups, composition components or method steps (or analogs or derivatives thereof as discussed above). Such additional structural groups, composition components or method steps, etc., however, do not materially affect the basic and novel characteristic(s) of the compositions or methods, compared to those of the corresponding compositions or methods disclosed herein. "Consisting essentially of or "consists essentially" or the like, when applied to methods and compositions encompassed by the present disclosure have the meaning ascribed in U.S. Patent law and the term is open-ended, allowing for the presence of more than that which is recited so long as basic or novel characteristics of that which is recited is not changed by the presence of more than that which is recited, but excludes prior art embodiments.

Prior to describing the various embodiments, the following definitions are provided and should be used unless otherwise indicated.

Definitions

In describing and claiming the disclosed subject matter, the following terminology will be used in accordance with the definitions set forth below.

The term "anesthesia" as used herein refers to a "reversible lack of awareness," whether this is a total lack of awareness (e.g. a general anesthetic) or a lack of awareness of a part of the body such as a spinal anesthetic or another nerve block would cause.

Anesthesia is a pharmacologically induced reversible state of amnesia, analgesia, loss of responsiveness, loss of skeletal muscle reflexes and decreased stress response.

The term "anesthetic (general)" is a drug that brings about a reversible loss of consciousness. These drugs are generally administered by an anesthesia provider in order to induce or maintain general anesthesia to facilitate surgery. Generally anesthetics, and in particular general anesthetics that typically induce a state of whole body unawareness, may be administered to a recipient animal or human as a gases or vapor (an inhalational anesthetic) or by injection, such as an intravenous anesthetics. In practice, it is common to administer both kinds to a single human patient, whereby an injection is given to induce anesthesia and a gas is used to maintain it, although it is possible to deliver anesthesia solely by inhalation or injection.

Inhalational anesthetic substances are either volatile liquids or gases and are usually delivered using an anesthesia machine. An anesthesia machine allows composing a mixture of oxygen, anesthetics and ambient air, delivering it to the patient and monitoring patient and machine parameters. Liquid anesthetics are vaporized in the machine.

Only a few inhalation anesthetics are in widespread use. Desflurane (suprane)

(difluoromethyl 1-fluoro-2,2,2-trifluoromethyl ether), isoflurane (1-chloro-2,2,2,-trifluoroethyl difluoromethyl ether) and sevoflurane (fluoromethyl 2,2,2,-trifluoro-1-[trifluoromethyl]ethyl ether) are the most widely used volatile anesthetics today and may be combined with nitrous oxide. Older, and less popular, volatile anaesthetics, include halothane (2-bromo-2-chloro- 1 ,1 ,-trifluoroethane), enflurane (ethrane) (2-chloro-1 ,1 ,2-trifluorethyl difluoromethyl ether), and methoxyflurane.

Injection anesthetics are used for induction and maintenance of a state of unconsciousness. Anesthetists prefer to use intravenous injections as they are faster, generally less painful and more reliable than intramuscular or subcutaneous injections. Among the most widely used drugs are: propofol (2,6-diisopropylphenol), etomidate, barbiturates such as methohexital and thiopentone/thiopental, benzodiazepines such as midazolam and diazepam, and ketamine.

The term "Na⁺-K⁺-2CI^" co-transporter 1 (NKCC1)" as used herein refers to a member of the class of proteins termed cation-chloride co-transporters (CCCs) that are important regulators of neuronal chloride concentration believed to influence cell-to-cell

communication, and various aspects of neuronal development, plasticity and trauma. The CCC gene family consists of three broad groups: Na⁺-CI^" co-transporters (NCCs), K⁺-CI^" co- transporters (KCCs) and Na⁺-K⁺-2CI^" co-transporters (NKCCs). Two NKCC isoforms have been identified: NKCC1 is found in a wide variety of secretory epithelia and non-epithelial cells, whereas NKCC2 is principally expressed in the kidney. There are two splice variants of the S1 c12a2 gene that encodes NKCC1 , referred to as NKCCI a and NKCC b. The NKCCIa gene has 27 exons, while the splice variant NKCCI b lacks exon 21. The NKCCI b splice variant is expressed primarily in the brain. NKCCI b is believed to be more than 10% more active than NKCCIa, although it is proportionally present in a much smaller amount in the brain than is NKCCIa. The differential splicing of the NKCC1 transcript may play a regulatory role in human tissues. The term "loop diuretic" as used herein refers to diuretics acting on the loop of Henle of the kidney and which inhibit the Na⁺-K⁺-2CI^" symport ion transporters. These inhibitors are a chemically diverse group including, but not limited to, furosemide, butmetanide (3- butylamino-4-phenoxy-5-sulfamoyl-benzoic acid), ethacrynic acid, benzmetanide, tripamide and the like, including all active pharmaceutically acceptable compounds of this description as well as various foreseen and readily provided complexes, derivatives, salts, solvates, isomers, enantiomers, polymorphs, and prodrugs of these compounds, and combinations thereof. .

The term "mineralcorticoid" as used herein refers to compounds that can bind to mineralcorticoid receptors (MR), thereby causing retention of salts and water, while increasing K⁺ and H⁺ excretion. In particular, the present disclosure incorporates the use of aldosterone receptor antagonists such as, but not limited to, spironolactone, such as 17- spironolactone, that blocks the effects of mineralcorticoids by acting as a receptor antagonist. Spironolactone can competitively inhibit the binding of aldosterone to an MR. Aldosterone receptor antagonists for use in the methods of the present disclosure are also intended to include all active pharmaceutically acceptable compounds of this description as well as various foreseen and readily provided complexes, derivatives, salts, solvates, isomers, enantiomers, polymorphs, and prodrugs of these compounds, and combinations thereof.

The term "oxytocin" as used herein refers to the mammalian hormone that acts primarily as a neuromodulator in the brain. Also known as a-hypophamine (a-hypophamine), oxytocin is a peptide of nine amino acids (a nonapeptide): its systematic name is cysteine- tyrosine-isoleucine-glutamine-asparagine-cysteine-proline-leucine-glycine-amine. The cysteine residues form a disulfide bond. The biologically active form of oxytocin is also known as the octapeptide "oxytocin disulfide" (oxidized form). The term "oxytocin" may further include, but is not limited to, analogs such as carbetocin, including all active pharmaceutically acceptable compounds of this description as well as various foreseen and readily provided complexes, derivatives, salts, solvates, isomers, enantiomers, polymorphs, and prodrugs of these compounds, and combinations thereof. Exemplary analogs for use in the methods of this disclosure include, but are not limited to, 4-threonine-1-hydroxy- deaminooxytocin, 9-deamidooxytocin, an analog of oxytocin containing a glycine residue in place of the glycinamide residue; 7-D-proline-oxytocin and its deamino analog; (2,4- diisoleucine)-oxytocin, an analog of oxytocin with natriuretic and diuretic activities; deamino oxytocin analog; a long-acting oxytocin (OT) analog, 1-deamino-1-monocarba-E12- [Tyr(OMe)]-OT(dCOMOT); carbetocin, (1-butanoic acid-2-(0-methyl-L-tyrosine)-1- carbaoxytocin, or, alternatively, deamino-1 monocarba-(2-0-methyltyrosine)-oxytocin

[d(COMOT)]); [Thr4-Gly7]-oxytocin (TG-OT); oxypressin; lle-conopressin; atosiban; deamino-6-carba-oxytoxin (dC60). It is further intended that the term "oxytocin" may include any oxytocin mimetic or small-molecule that can specifically recognize the oxytocin receptor, and thereby induce receptor-initiated signaling similar to when oxytocin specifically binds to the receptor.

The term "side-effect" as used herein refers to a physiological, neurological, or biochemical change in an animal or human subject that has received an amount of an anesthetic, and wherein the effect is other than the induction of an anesthetic response. Such side-effects include, but are not limited to, decreases in blood pressure (hypotension), tachycardia, changes in respiratory rate, cerebral vascular flow, seizures, memory loss, and the like.

The term "neural activity" as used herein refers to a physiological, neurological, or biochemical change in an animal or human subject derived from the activity of the cells of the neural system (for example, but not limited to, the central nervous system including the brain, brain stem, and peripheral neural network), and wherein the effect is other than the induction of an anesthetic response. Such neural activites include, but are not limited to, induction of a seizure, a neurotoxicity, a behavioral effect, a cognitive effect, or any combination thereof, decreases in blood pressure (hypotension), tachycardia, changes in respiratory rate, cerebral vascular flow, memory loss, and the like.

The term "agonist" as used herein refers to a compound or molecule, including but not limited to, peptides, oligopeptides, and small molecules, variants and derivatives thereof that may interact with a receptor of a cell, thereby inducing an increase in a biochemical or physiological activity attributable to the receptor. The agonist may be, but is not limited to, a natural ligand effector of the receptor, an analog or a mimetic and the like thereof.

The term "antagonist" as used herein refers to a compound or molecule, including but not limited to, peptides, oligopeptides, and small molecules, variants and derivatives thereof that may interact with a receptor of a cell, thereby inducing a decrease in a biochemical or physiological activity attributable to the receptor. The agonist may be, but is not limited to, a natural inhibitor of the receptor, an analog or a mimetic and the like thereof.

The terms "subject" and "subject animal or human" as used herein refers to any animal, including a human, to which a composition according to the disclosure may be delivered or administered.

Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art of molecular biology. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, suitable methods and materials are described herein. Further definitions are provided in context below. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art of molecular biology. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, suitable methods and materials are described herein.

Description

Increasing numbers of preterm and critically ill term neonates and infants survive due to advances of modern medicine. Care for these and other patients of this age category, however, often involves both surgical and non-surgical procedures that require short-term or prolonged administration of anesthesia (Rose VL. (1999) Am. Fam. Physician 59: 2925; Hall RT. (2000) Pediatrics 105: 1 137-1 140).

Animal data showing cytotoxic and delayed behavioral effects of neonatal anesthesia in combination with numerous clinical reports of anesthesia-associated paradoxical hyperexcitatory events (see, for example) Satomoto ef a/., (2009) Anesthesiology 1 0: 628- 637; Stratmann ef a/., (2009) Anesthesiology 1 10: 834-848; 849-861 ; Zhen ef a/., (2009)

Anesthesiology 1 1 1 : 741 -752; Zhang ef a/., (2008) J. Neurosci. 28: 4551 -4560; Kahraman ef a/., (2008) J. Neurosurg. Anesthesiol. 20: 233-240; Zhang et al., (2008) Neurosci. Lett. 447:109-1 14; Ma et al., (2007) Anesthesiology^: 746-753; Yon et al., (2005)

Neuroscience. 135: 815-827; Jevtovic-Todorovic et al., (2003) J. Neurosci. 23: 876-882; Perouansky et al., (2009) Anesthesiology 1 1 1 : 1365-1371 ; Zeiler ef al, (2008) Seizure 17: 665-667; Welborn et al., (1996) Anesth. Analg. 183: 917-920; Cravero et al., (2000) Paediatr. Anaesth. 10: 4 9-424; Veyckemans F. (2001 ) Curr. Opin. Anaesthesiol. 14: 339- 343; Constant ef al., Paediatr. Anaesth. 15: 266-274; Hickey ef a/., (2005) J. Emerg. Med. 29: 447-449; Cohendy ef al., (2005) Curr. Opin. Clin. Nutr. Metab. Care. 8: 17-21 ; Mohanram ef al., (2007) Can. J. Anaesth. 54: 657-66 ; Singh ef al., (2007) Anesth. Analg. 104: 233- 234; Griffeth & Mehra J. ECT. 23: 177-1778; Harrison JL. (1986) Anesth. Analg. 65: 1235- 1236; McManus KF. (1992) Anaesth. Intensive Care 20: 245) and with retrospective epidemiological studies suggesting a link between anesthesia and later learning defects (DiMaggio et al., (2009) J. Neurosurg. Anesthesiol. 21 : 286-291 ; Wilder et al., (2009) Anesthesiology 1 10: 796-804) raise tremendous concerns about the safety of general anesthesia at early stages of brain development (Perouansky &Hemmings (2009)

Anesthesiology 1 1 1 : 1365-1367). Although, the types and frequency of anesthesia-related deficits and their underlying mechanisms remain poorly studied, the alteration of neurogenesis caused by anesthetic-depressed neuronal activity has been a predominant concept (Yon et al., (2005) Neuroscience 135: 815-827; Jevtovic-Todorovic et al., (2003) J. Neurosci. 23: 876-882). As described in the present disclosure, it has now been found that anesthesia of neonatal rats with the volatile anesthetic sevoflurane, the most widely used general anesthetic in pediatric patients, can cause cortical seizures and cytotoxicity; later on, when these animals achieved juvenile age, impairment in synaptic plasticity, sensorimotor gating function and grooming behavior can be detected. Bumetanide, a specific inhibitor of the Na⁺-K⁺-2CI^" importer (NKCC1 ), administered prior to sevoflurane, alleviated most of these adverse effects. The data indicate that isoflurane may differ in the induction of cortical seizures but may also cause bumetanide-responsive cytotoxicity and impairment in sensorimotor gating function.

Due to high N KCC1 activity, immature neurons have significantly elevated intracellular concentrations of CI^". Activation of γ-aminobutyric acid type A (GABA_A) and strychnine-sensitive glycine receptors in these cells results in CI^" efflux, membrane depolarization (see, for example Wilder et al., (2009) Anesthesiology 1 10: 796-804; Edwards ef al., (2010) Anesthesiology; Pfeffer ef al., (2009) J Neurosci. 2009; 29: 3419-3430; Ye JH . (2008) Results Probl Cell Differ. 44: 123-143; Ben-Ari Y. (2002) Nat. Rev. Neurosci. 3: 728- 739; Staley er a/., (1995) Science 269: 977-981 ), activation of voltage-gated Na⁺ and Ca⁺⁺ channels and relief of the Mg⁺⁺-block of Ca⁺⁺ permeable N-methyl-D-aspartate (N MDA) receptors (see, for example Wilder ef al., (2009) Anesthesiology 1 1 0: 796-804; Edwards ef al., (2010) Anesthesiology; Pfeffer et al, (2009) J Neurosci. 2009; 29: 341 9-3430; Ye JH . (2008) Results Probl Cell Differ. 44: 123-143; Ben-Ari Y. (2002) Nat. Rev. Neurosci. 3: 728- 739; Staley ef al., (1995) Science 269: 977-981 ; Yamada et al., (2004) J. Physiol. 557: 829- 841 ; Owens et al., (1 996) J Neurosci. 16: 6414-6423). GABA-activated Ca⁺⁺ influxes play a crucial role in modulation of practically all elements of brain development, from neuronal progenitor proliferation to formation of new synapses (see, for example Wilder ef al., (2009) Anesthesiology 0: 796-804; Pfeffer ef al., (2009) J Neurosci. 2009; 29: 3419-3430;

Yamada ef al., (2004) J. Physiol. 557: 829-841 ; Owens ef al., (1996) J Neurosci. 16: 6414- 6423). Therefore, anesthetics that enhance GABA_A/glycine receptor activity may exacerbate GABA-induced depolarization and Ca²⁺ influx in immature neurons resulting in immediate (seizures, cytotoxicity) and delayed behavioral/cognitive defects.

Accordingly, the present disclosure encompasses methods of reducing the side- effects of an anesthetic, and in particular a reduction in the induction of seizures in the brains of mammals exposed to an anesthetic. Accordingly, data is provided that the administration of a Na⁺-K⁺-2CI^" co-transporter 1 inhibitor before, or simultaneously with , but not after, the anesthetic results in a diminution of seizures, neurotoxicity and/or impairment in synaptic plasticity effects. In particular, the methods of the disclosure encompass the administration to a subject animal or human of a therapeutic compound including a loop diuretic such as, but not limited to, bumetanide, a mineralcoricoid receptor antagonist, or oxytocin (or a therapeutically effective variant thereof) before or with an anesthetic such as isoflurane, sevoflurane, and the like, and whereby the administered therapeutic agent can reduce or eliminate at least one side-effect of the anesthetic subsequently presented to the recipient animal or human subject.

In the experiments disclosed herein, for example, electroencephalography, activated caspase-3, and hippocampal long-term potentiation were measured in rats exposed to 2.1% sevoflurane for 0.5-6 hrs at postnatal days 4-17 (P4-P17). Arterial blood gas samples drawn at a sevoflurane concentration of 2.1 % showed no evidence of either hypoxia or

hypoventilation in spontaneously breathing animals. Higher doses of sevoflurane (e.g. 2.9%), however, caused respiratory depression. During anesthesia maintenance, electroencephalographies demonstrated exhibited distinctive episodes of epileptic seizures in 40% of P4-P8 rats. Such seizure-like activity was not detected during anesthesia maintenance in older P10-P17 rats.

Emergence from 3 hrs of anesthesia with sevoflurane resulted in tonic/clonic seizures in some P10-P17 rats, but not in P4-P8 rats. Bumetanide (5 pmol/kg, administered intraperitoneally), significantly decreased seizures in P4-P9 rats, but did not affect the emergence seizures in P10-P17 rats. Anesthesia of P4 rats with sevoflurane for 6 hours caused a significant increase in activated caspase-3, and impairment of long-term potentiation induction, measured 1 and 14-17 days after exposure to sevoflurane, respectively. It was found, however, that pretreatment of P4 rats with bumetanide nearly abolished the increase in activated caspase-3, but did not alleviate impairment of long-term potentiation.

While not wishing to be bound by any one theory, it is contemplated that a general anesthesia such as with sevoflurane can cause epileptic seizures and neurotoxicity in neonatal rats, and that these effects can be diminished by early administration of the NKCC1 transporter inhibitor bumetanide. Bumetanide did not affect emergence seizures in older rat pups, indicating different underlying mechanisms in these two types of epileptic seizures associated with sevoflurane anesthesia. Also, an impairment of hippocampal long-term potentiation could be detected weeks after sevoflurane anesthesia in neonatal rats, an effect that was not significantly ameliorated by pretreatment with bumetanide.

Seizure-like phenomena related to anesthesia have been reported in human patients of all ages from neonates through those in their eighties, though more frequently during early periods of life. Many of these seizures do not have a typical clinical phenotype and would only be detected by electroencephalographic monitoring, which is not routinely done.

Therefore, the true incidence of seizures caused by anesthetics, particularly in the youngest patients, who are vulnerable to injury to their developing brains, is not known but may be quite high even in the absence of central nervous system diseases or severe systemic illness.

Although, a wide range of cellular mechanisms mediate the anesthetic action of sevoflurane, GABA_A/glycine receptor-mediated CI^" currents appear to be a likely mechanism to contribute to the excitatory effect of sevoflurane at early stages of brain development. The concentrations of bumetanide of this study that counteracted the seizure-induction of sevoflurane selectively inhibit NKCC1 , indicate that the inhibition of CI^" accumulation is the most likely mechanism by which bumetanide exerts its inhibition of the epileptogenic and neurotoxic effects of sevoflurane. Conversely, these data are consistent with the possibility that sevoflurane causes its epileptogenic and neurotoxic effects in the developing brain, at least in part, by potentiating GABA_A/glycine receptor-mediated depolarizing CI^" currents.

Bumetanide's well known renal diuretic effect is an unlikely mechanism to explain these results. There was no obvious increase in urine production in the subject rat pups after bumetanide administration, possibly because such an observation is difficult given the very small body size of the rat pups, or because of the immature renal function at this age. In a human neonate, bumetanide exerted antiepileptic effects during 2 hrs of observation following a single dose with only slightly increased urine output, (Kahle er a/., (2009) J. Child Neurol. 24: 572-576). If a non-specific electrolyte mediated effect on seizure threshold existed, then bumetanide should have also decreased emergence seizures in older rat pups, an effect not observed.

The blood gas analyses performed in the control animals lend further support that the observed adverse effects are specific effects of the anesthetic and not related to

pertubations caused by the experimental manipulations. Consistent with the effects of bumetanide is data that bumetanide decreased GABA-mediated depolarization in immature neurons, and cortical seizure activity in neonatal rats both in vitro and in vivo. NKCC1 - mediated actions of bumetanide are also supported by findings that NKCC1 -mediated effects are not observed in the presence of antagonists of the GABA_A receptors and that bumetanide did not affect epileptiform activity in brain slices from NKCC1-/- mice. The assumption that a single dose of bumetanide achieves a sufficient effect in the central nervous system is likely based on its lipid/water solubility and on the slow elimination of bumetanide in neonatal patients. Pharmacokinetic studies of bumetanide have not been performed in neonatal rats. However, in preterm and full-term human neonates, bumetanide is reported to have a half life of approximately 6 hours, with a range up to 15 hours (Eades & Christensen (1998) Pediatr. Nephrol. 12: 603-616).

Experimental data on neuronal death in neonatal rodents caused by other anesthetics with a GABA_A/glycine-ergic component of action also indirectly support a role of excitatory mechanisms in these effects. Thus, 6 hr long sedation with isoflurane caused significant neurodegeneration in P7 rat pups (Jevtovis-Todorovic ef a/., (2003) J. Neurosci. 23: 876-882). Another study reported that P7 rats, treated for 6 h with isoflurane exhibited significant and dose-dependent neurodegeneration, while P10 and P14 rats showed no significant increase in apoptotic neurodegeneration. The P5-P7 period coincides with the peak expression of NKCC1 and peak [Cl^"]|. Therefore, an increase in neuronal activity at this developmental stage in the presence of isoflurane, whose mechanisms of action include activation of GABA_A/glycine receptors is plausible. In further support of this possibility, GABA activation in newborn rats caused excessive calcium influx and cell death (Nunez & McCarthy (2003) Exp. Neurol. 181 : 258-269). In addition, an in vitro study indicated that propofol increased intracellular Ca⁺⁺ concentration and neurotoxicity in cultured hippocampal neurons in vitro at day 4 but not day 8 (Kahraman et a/., (2008) J. Neurol. Anesthesiol. 20: 233-240). Sevoflurane has also been shown to cause apoptosis and learning deficits and abnormal social behaviors in mice exposed to sevoflurane at P6-P7 (Zhang er a/. , (2008) Neurosci. Letts. 447: 109-1 14; Satomoto et a/. , (2009) Anesthesiology 1 10: 628-637).

Similar to rat pups, humans show high neuronal expression of NKCC1 and low expression of KCC2 before the end of the first year of life indicating that the results of the present disclosure are applicable to human premies and neonates. The developmental shift in the expression ratio of NKCC1/KCC2 occurs sooner in female than male rats (Lemonnier & Ben-Ari (2010) Acta Paediatr. 99: 1885-1888). If similar differences in the developmental shift in expression ratio of NKCC1 /KCC2 takes place in humans, neonate and infant male patients may be more prone to adverse effects of GABA_A/glycine receptor enhancing anesthetics.

While not wishing to be bound by any one theory, it is possible that despite the lack of efficacy of bumetanide, GABA_A/glycine receptor-mediated inhibition produced by sevoflurane may play a role in the emergence seizures observed in rats with a mature neuronal CI^" gradient. Prolonged and/or enhanced inhibition produced by sevoflurane anesthesia via all mechanisms whereby sevoflurane acts, including sevoflurane-caused depression of glutamate release, may initiate compensatory increases in excitatory output to balance enhanced inhibition. Abrupt withdrawal of sevoflurane and decreased inhibition shifts the inhibition/excitation balance towards greater excitation (e.g., increased glutamate release and activation of N-methyl D-aspartate receptor- and voltage-operated Ca⁺⁺ channels). Increased intracellular Ca⁺⁺ may increase inhibition of KCC2 and shift E in mature neurons to more positive values. Both an increase in excitatory glutamatergic output, and excitatory or less than normal inhibitory output, of GABA and glycine systems due to inhibited activity of KCC2 may provide a basis for the observed hyperexcitability phenomena during emergence. Furthermore, if Ε_α is indeed shifted to more positive values, agents with predominately GABA_A/glycine receptor-mediated activity such as benzodiazepines may be ineffective in treating emergence hyperexcitability phenomena. In contrast, anesthetics with predominately an anti-glutamatergic action such as ketamine may in fact be the drugs of choice for treating emergence hyperexcitation. In support of these hypotheses, are clinical data that anesthetics with anti-NMDA actions relieve emergence hyperexcitability in patients, whereas midazolam, which enhances GABA-ergic signals, does not.

The acute effects of anesthesia were assessed by measuring

electroencephalographic (EEG) activity in the sevoflurane-anesthetized postnatal days 4-9 (P4-P9) rats. To study the delayed effects, all rats were anesthetized at P4-P5 and activated caspase-3 levels were evaluated either two hours or one day later while prepulse inhibition (PPI) of acoustic startle response and grooming behavior were evaluated at P24. All treatments were administered either prior to or during the administration of sevoflurane.

A developmental pattern of action in the brain may also be applicable to the mineralocorticoid hormone, aldosterone that may participate in mediation and, at abnormal levels, exacerbation of the side effects associated with neonatal sevoflurane anesthesia. A number of factors suggest that aldosterone exerts much stronger effects in the brain in the early stages of life. Strong expression of HSD2 in the embryo and early postnatal brain that almost disappears in most of the brain regions soon after birth provides conditions for effective interaction of aldosterone with its MRs at early stages of brain development (Geerling & Loewy (2009) Am. J. Physiol. Renal Physiol. 297: F559-576). Such interaction is even more likely due to the fact that newborn infants have significantly higher levels of aldosterone (Martinerie et a/. , (2009) Pediatr Res. 66: 323-328). Furthermore, aldosterone levels are higher in preterm infants than in full-term infants (Nader et al. , (1996) J. Pediatr. (Rio J.) 72: 143-150; Semama et al. , (2007) Arch. Pediatr. 14: 249-253; Mildenberger & Versmold (2002) Eur. J. Pediatr. 161 : 415-422). Similar to neuronal CI^" gradient maturation that determines excitatory/inhibitory output of GABA_A receptor activation (Dzhala et al. , (2005) Nat. Med. 1 1 : 1205-1213), aldosterone levels decrease to adult levels during the first year of life (Mehta et al., (1992) Indian Pediatr. 29: 1385-1390). Although aldosterone may be synthesized in the brain, the adrenal glands are likely the main source of aldosterone in the brain (Connell & Davies (2005) J. Endocrinol. 186: 1 -20). Therefore, the blood-brain barrier is an important determinant of aldosterone levels in the brain. Given the fact that the blood-brain barrier is not completely formed in neonates and that the general anesthesia may further compromise functioning of the blood-brain barrier, the impact of aldosterone action on the brain during general anesthesia can be even stronger, especially in preterm infants.

Finally, general anesthesia and opioids stimulate the release of aldosterone making aldosterone an anesthesia-specific messenger (Norberg ef al. , (2007) Anesthesiology 107: 24-32; Koda er a/., (2005) J. Clin. Anesth. 17: 3-7; Petropoulos er a/., (2000) Clin. Exp. Obstet. Gynecol. 27: 42-46; Lesage et al., (2000) Life Sci. 66: 197-121 1 ). The anesthesia- stimulated release of aldosterone may be greater in the developing brain due to the depolarizing action of GABA. In addition, sevoflurane-induced activation of two-domain potassium leak channels, and following increase of extracellular K⁺, may also lead to increase of aldosterone release. In agreement with the developmental profile of aldosterone action in the brain are the results of the present disclosure that aldosterone enhanced cortical seizures in P4-P6 rats, but did not alter the EEG activity of P17-P20 rats during anesthesia with sevoflurane. Whether aldosterone increases cortical seizures, levels of activated caspase-3 and disruption of PPI in sevoflurane-anesthetized neonatal rats by enhancing the NKCC1 activity is not understood. Bumetanide does not prevent cortical seizures enhanced by aldosterone, and, in some animals, even enhanced them, supports another mechanism of action of aldosterone.

While not wishing to be held to any one theory, bumetanide likely depresses NKCC1 activity and increases the release of aldosterone (Haloui et a/., (2001 ) Cardiovasc. Res. 5 : 542-552), promoting non-NKCC1 action of aldosterone on EEG activity during sevoflurane anesthesia. Also aldosterone did not affect grooming behavior in the sevoflurane- anesthetized rats, which was significantly depressed by pretreatment with bumetanide. However, since high levels of aldosterone are associated with hyperkalemia (Martinerie ef a/., (2009) Pediatr. Res. 66: 323-328; Nader ef a/., (1996) J. Pediatr. (Rio J.) 72: 143-150; Semama et a/. , (2007) Arch. Pediatr. 14: 249-253; Mildenberger ef a/. , (2002) Eur. J.

Pediatr. 161 : 415-422) potassium-enhanced activity of NKCC1 may contribute to the aldosterone-induced side effects (Fig. 10). According to the experiments with

spironolactone, which normalized levels of activated caspase-3 and PPI of startle in sevoflurane-anesthetized rats, but did not antagonize cortical seizures during sevoflurane anesthesia and sevoflurane-altered grooming behavior, endogenous aldosterone may be involved in the mediation of some, but not all, side effects of sevoflurane in otherwise healthy neonatal rats. The role of endogenous aldosterone in the side effects caused by sevoflurane is also indirectly supported by exogenous aldosterone affecting the levels of activated caspase-3 and PPI of startle only in the sevoflurane-anesthetized animals, pointing to the possibility that endogenous and exogenous aldosterone may have an additive effect.

Spironolactone alone increased levels of activated caspase-3 and disruption of PPI of startle and time spent grooming in non-anesthetized rats, though not as strongly as sevoflurane. These actions of spironolactone, among others, may involve the NKCC1 - mediated component. By displacing aldosterone from MRs, spironolactone increases levels of aldosterone and extracellular K⁺. Both extracellular K⁺ and aldosterone provided this action of aldosterone does not involve activation of MRs, may stimulate NKCC1 activity. In rat ventricular myocytes, spironolactone - similar to aldosterone - enhanced intracellular Na⁺ and volume, while these effects of aldosterone were depressed by bumetanide (Matsui er a/. , (2007) Can. J. Physiol. Pharmacol. 85: 264-273). The NKCC1 - dependent action of spironolactone may be responsible, at least for some increase in time spent grooming by the spironolactone-pretreated, non-anesthetized rats. Bumetanide normalized sevoflurane-altered grooming behavior, while aldosterone did not have significant effect on time spent grooming, indicating that the sevoflurane-induced changes in PPI of startle and grooming behavior may be mediated by different mechanisms.

Given that spironolactone did not depress the sevoflurane-caused EEG seizures but normalized the PPI of startle and levels of activated caspase-3 altered by sevoflurane, it is possible that the cortical seizures are not the necessary element in sevoflurane action to cause delayed neurotoxic and behavioral abnormalities. Spironolactone may depress activation of caspase-3 and normalize PPI of startle in the sevoflurane anesthetized animals by depressing non-NKCC1 action of aldosterone.

Oxytocin, or carbetocin, a synthetic analog of oxytocin with a substantially longer elimination half-life depressed cortical seizures during sevoflurane anesthesia, decreased grooming behavior, and improved PPI of startle. These effects are in agreement with the known biological properties of oxytocin. Thus, oxytocin may inhibit the NKCC1 activity in immature neurons that results in switching of GABA signaling from excitatory to inhibitory, rendering GABA action neuroprotective (Tyzio ef a/., (2006) Science 314: 1788-1792). Also, oxytocin was shown to exert anxiolytic, antidepressant, and anti-inflammatory effects, and other actions (Ring ef a/., (2010) Neuropharmacology 58: 69-77; Welch ef a/. , (2010) Neurogastroenterol. Motil. 22: 654-e202). The neural effects of oxytocin and agonists of its receptors have been intensively studied due to their potential to be used in therapy for a variety of human psychiatric diseases, including anxiety disorders, autistic spectrum disorders, and schizophrenia.

Carbetocin may be more effective than bumetanide in diminishing the adverse effects of neonatal anesthesia with sevoflurane. Its effectiveness may be that activation of oxytocin receptors not only diminishes the excitatory output of GABA_A receptor-mediated signaling, but also initiates anti-inflammatory responses that may antagonize the inflammatory action of aldosterone, as shown in Fig. 20. Thus, the side effects caused by sevoflurane in neonatal rats are likely due in part to an increase in the aldosterone level, and to decreased levels of endogenous oxytocin during anesthesia. For instance, elevated aldosterone induced by anesthesia, acting via negative feedback on rennin-angiotensin system, may decrease the level of angiotensin IV. Lower concentrations of angiotensin IV lead to disinhibition of the constitutive peptidase activity of AT4 receptors, resulting in a decrease of oxytocin levels. Altogether, the results suggest that sevoflurane anesthesia-induced side effects may be even more intense in subjects that have abnormally functioning aldosterone and oxytocin systems prior to anesthesia. The potency of aldosterone action in the brain is expected to be at its peak during the critical perinatal period, and, as it appears from this study, may be further strengthened during general anesthesia. Given the systemic action of aldosterone, neonatal anesthesia may have adverse effects not only on brain-related functions traditionally studied in animals, such as behavior, but also profound systemic adverse effects in which aldosterone system plays an important role, such as metabolic disorders. The agents that affect the aldosterone and oxytocin systems therefore can be effective tools for improving the efficacy and safety of neonatal anesthesia.

It is further contemplated that the disclosure encompasses embodiments of methods for reducing the intensity of seizures, including epileptogenic seizures that may or may not be the result of the administration of an anesthetic to a subject animal or human. It has been shown that oxytocin and at least one aldosterone receptor antagonist are effective in reducing the level of intensity of at least one side-effect from an anesthetic and it is contemplated that such agents are effective also in reducing the onset or duration of a non- anesthetic induced seizure.

In summary, therefore, anesthesia of neonatal rats with sevoflurane is associated with episodes of cortical hyperexcitation (cortical seizures or electroclinical dissociation) and increased levels of activated caspase-3 in the brain tissue. Both EEG-detectable seizures in the presence of sevoflurane and increased levels of activated caspase-3 after exposure of neonatal rats to sevoflurane were sensitive to pretreatment with the loop diuretic, bumetanide. When used at relatively low doses, bumetanide is a specific inhibitor of the Na⁺-K⁺-2CI^" co-transporter (NKCC1 ) in neurons. High expression of NKCC1 in late embryonic and early neonatal cortical neurons is responsible for the elevated levels of intracellular CI^", and provides the basis for the depolarizing and excitatory actions of GABA, otherwise a major inhibitory neurotransmitter in the central nervous system. The GABA- induced depolarization can be excitatory, though it frequently causes inhibition because of shunting of the ion conductance with a more positive reversal potential. The bumetanide- induced depression of EEG seizures and apoptosis in the sevoflurane-anesthetized neonatal rats suggests that sevoflurane, by enhancing GABA_A receptor-mediated depolarizing CI^" conductance, increases the probability of the excitatory output of GABA_A receptor activation which, in turn, can contribute to the observed abnormalities. At least two actions of sevoflurane as an anesthetic may lead to increase of GABA_A receptor-mediated excitation. Besides direct enhancement of GABA_A receptor activity, the long-lasting activation of two domain potassium leak channels by sevoflurane may result in depletion of intracellular K⁺ and increase of extracellular K⁺, which can lead to stimulation of NKCC1 activity. Stimulation of the NKCC1 -mediated CI^" import should ultimately result in shift of the equilibrium potential for CI^" to more depolarizing values.

The excitatory output of GABA_A receptor activation may be further increased by a steroid hormone of the mineralocorticoid family, aldosterone, which, as was shown in nonneuronal cells, can increase NKCC1 activity. General anesthesia is associated with increased levels of aldosterone in the body (Norberg et al. (2007) Anesthesiology 107: 24- 32; Koda er a/., (2005) J. Clin. Anesth. 17: 3-7; Petropoulos er a/. , (2000) Clin. Exp. Obstet. Gynecol. 27: 42-46; Oyama et al (1979) Br. J. Anaesth. 51 : 747-752). The release of aldosterone during general anesthesia may not be specifically directed to the developing brain, per se, but availability of aldosterone (mineralocorticoid) receptors (MRs) for binding with aldosterone is high in fetal and early postnatal brain, and gradually decreases afterwards. This is because glucocorticoids, which are found in the brain in much higher concentrations than aldosterone, compete with aldosterone for MRs, and thus shield the MRs from activation by aldosterone. The enzyme, 1 1-β-hydroxysteroid dehydrogenize 2 (HSD2), by oxidizing glucocorticoids, increases MR availability for binding with aldosterone. The expression of HSD2 in the brain is relatively high in embryos and neonatal animals, but mostly disappears shortly after the birth, suggesting that the brain during the perinatal period is especially vulnerable to excess of aldosterone. Besides stimulation of the NKCC1 activity, aldosterone is known to cause a number of adverse effects on its own, such as cell inflammation, hypocalcemia, hypomagnesaemia that can lead to apoptosis, seizures, and cognitive deficiency (Tirosh et al., (2010) Curr. Hypertens. Rep. 12: 252-257; Gilbert & Brown (2010) Curr. Opin. Endocrinol. Diabetes Obes. 17: 199-204; Yagi ef al., (201 1 ) Hypertens. Res. 34: 74-78).

In contrast to aldosterone, nonapeptide hormone oxytocin depresses the NKCC1 activity (Tyzio et al., (2006) Science 314: 1788-1792) while oxytocin levels can be decreased during general anesthesia, especially in the presence of opioids. Oxytocin causes a number of other effects opposite to those produced by aldosterone, such as anti-inflammatory, anxiolytic, and antidepressant actions, indicating that the excitatory action of sevoflurane in combination with anesthesia-induced changes in the levels of aldosterone and oxytocin may be important contributing factors to abnormalities associated with neonatal anesthesia. If so, neonatal anesthesia-caused defects can be even more severe in cases of preexisting abnormalities in the aldosterone and oxytocin systems that are characterized by increased and decreased levels of aldosterone and oxytocin, respectively.

Sevoflurane, in addition to previously reported seizure-like activity and increased levels of activated caspase-3, caused impairment of PPI of startle and increase of time spent in non-syntactic chain grooming; all effects were alleviated by pretreatment with bumetanide (5 pmol/kg, i.p.)., a specific inhibitor of the Na⁺-K⁺-2CI^" co-transporter. Bumetanide without anesthesia did not affect either PPI of startle or grooming behavior. Aldosterone receptor antagonist, spironolactone (20 mg/kg, s.c), normalized activated caspase-3 levels and PPI of startle altered by sevoflurane, but was not effective in preventing cortical seizures.

Exogenous aldosterone (20 mg/kg, s.c.) further increased cortical seizures, level of activated caspase-3 and disruption of PPI of startle, but not grooming. Cortical seizures in the presence of aldosterone were not responsive to pretreatment with bumetanide. Aldosterone has no effect on EEG activity of P17-P21 rats. Oxytocin or its synthetic analog, carbetocin (1.5 pg, i.c.v.), depressed sevoflurane-caused cortical seizures and normalized PPI of startle and grooming behavior.

These findings indicate: 1 ) neonatal anesthesia with sevoflurane may cause brain function abnormalities that are hallmarks of major neuropsychiatric disorders; 2) excitatory action of GABA and enhanced action of aldosterone in the developing brain as well as lower levels of oxytocin may play important roles in these complications; 3) preexisting abnormalities in aldosterone and oxytocin systems may exacerbate complications caused by neonatal sevoflurane.

Accordingly, while sevoflurane anesthesia in the early postnatal period may result in both acute and delayed abnormalities in brain functioning; these abnormalities can be effectively diminished by supplementing sevoflurane exposure with bumetanide, a loop diuretic that inhibits the NKCC1 co-transporter. While the mineralocorticoid hormone, aldosterone, exacerbated the adverse effects of sevoflurane in neonatal rats, the side effects caused by sevoflurane were also diminished by the aldosterone receptor inhibitor, spironolactone, and the polypeptide hormone, oxytocin, or its synthetic analog, carbetocin.

sevoflurane.

In embodiments of this aspect of the disclosure, the anesthetic increases

GABA_A/glycine receptor activity in the recipient subject animal or human.

In embodiments of this aspect of the disclosure, the oxytocin receptor agonist can be selected from the group consisting of: oxytocin, carbetocin, or an analog or a derivative thereof. Yet another aspect of the disclosure encompasses embodiments of a method of determining the likelihood of an animal or human subject developing a side-effect of an anesthetic administered to said subject, the method comprising determining whether the subject has an abnormality in a level of aldosterone or oxytocin compared to a normal level, or an abnormality in the physiological function or response thereto of aldosterone or oxytocin in the animal or human subject, wherein the presence of the abnormality indicates the likelihood of an animal or human subject developing a side-effect of an anesthetic administered to said subject.

The specific examples below are to be construed as merely illustrative, and not limitative of the remainder of the disclosure in any way whatsoever. Without further elaboration, it is believed that one skilled in the art can, based on the description herein, utilize the present disclosure to its fullest extent. All publications recited herein are hereby incorporated by reference in their entirety.

It should be emphasized that the embodiments of the present disclosure, particularly, any "preferred" embodiments, are merely possible examples of the implementations, merely set forth for a clear understanding of the principles of the disclosure. Many variations and modifications may be made to the above-described embodiment(s) of the disclosure without departing substantially from the spirit and principles of the disclosure. All such modifications and variations are intended to be included herein within the scope of this disclosure, and the present disclosure and protected by the following claims.

The following examples are put forth so as to provide those of ordinary skill in the art with a complete disclosure and description of how to perform the methods and use the compositions and compounds disclosed and claimed herein. Efforts have been made to ensure accuracy with respect to numbers (e.g., amounts, temperature, etc.), but some errors and deviations should be accounted for. Unless indicated otherwise, parts are parts by weight, temperature is in °C, and pressure is at or near atmospheric. Standard temperature and pressure are defined as 20 °C and 1 atmosphere.

It should be noted that ratios, concentrations, amounts, and other numerical data may be expressed herein in a range format. It is to be understood that such a range format is used for convenience and brevity, and thus, should be interpreted in a flexible manner to include not only the numerical values explicitly recited as the limits of the range, but also to include all the individual numerical values or sub-ranges encompassed within that range as if each numerical value and sub-range is explicitly recited. To illustrate, a concentration range of "about 0.1 % to about 5%" should be interpreted to include not only the explicitly recited concentration of about 0.1 wt% to about 5 wt%, but also include individual concentrations (e.g., 1 %, 2%, 3%, and 4%) and the sub-ranges (e.g., 0.5%, 1 .1 %, 2.2%, 3.3%, and 4.4%) within the indicated range. The term "about" can include ±1 %, ±2%, ±3%, ±4%, ±5%, ±6%, ±7%, ±8%, ±9%, or ±10%, or more of the numerical value(s) being modified.

EXAMPLES

Example 1

Animals: Sprague Dawley rats were studied. To control for litter variability, several pups were used for each treatment condition from each litter. At the beginning of each experiment, the pups were determined to be well nourished, judged by their stomachs being full of milk (detectable through the transparent abdominal wall).

Example 2

Anesthesia and electroencephalogram recording: To determine the effects of sevoflurane on cortical activity, postnatal days 4 to 20 (P4-P20) rat pups were instrumented for

electroencephalogram recording. Half of the P5 and earlier rats were anesthetized by cold immersion (to avoid additional exposure to anesthetic); the remaining half and older rats were anesthetized by inhalation anesthesia with isoflurane (1 .8-2.5%).

Using bregma as needed for a reference, four holes (0.5 mm) were burred (Microtorque

II, WPI, Sarasota, FL) in the skull at bilateral frontal and occipital regions for implantation of 4 electrodes of the headmounts of the electroencephalogram/electromyogram system (Pinnacle Technology, Lawrence, KS) (as shown in Fig. 1 A). The entire instrumentation for

electroencephalogram recording took no more than 20 min. Electroencephalogram recording started immediately after completion of the implant surgery and ended 30 min after termination of anesthesia with sevoflurane. The 60 min anesthesia-free interval after the implant surgery before exposure to sevoflurane allowed recording of a baseline electroencephalogram.

Sevoflurane (Fushimi-machi, Osaka, Japan) anesthesia was induced with 6% sevoflurane and 1 .5 L/min oxygen over 3 min, and maintained with 2.1 % sevoflurane and 1 .5 L/min oxygen over 30-360 min in a thermostated chamber. Backdraft through wall vacuum was used to scavenge waste gases. Onset and offset of anesthesia were monitored via electroencephalogram and by loss and return of righting reflex, respectively. Anesthesia gas monitoring was performed using a calibrated Datex side stream analyzer that sampled from the interior of the animal chamber. To assure adequate oxygenation and respiration, some of the rat pups were anesthetized with the same sevoflurane protocol as rats in the study group and arterial blood gas and glucose determinations were performed by cardiac puncture at the conclusion of the anesthetic. Arterial blood gases and glucose were measured using a portable clinical analyzer (i-STAT, Abbott Laboratories Inc., East Windsor, NJ).

Continuous electroencephalogram recordings from bilateral occipital and right frontal regions in rat pups were performed using an electroencephalogram/electromyogram system (Pinnacle Technology). Acquisition of the electroencephalogram was performed using the Sirenia software (Pinnacle Technology). Sampling interval per signal was 200 ps (5 kHz). Sirenia Score (Pinnacle Technology) and Clampfit 9.2 (Axon Instruments, Union City, CA) programs were used for the electroencephalogram data analysis. Data were filtered offline using a bandpass Bessel (8-pole) 0.04Hz-56 Hz filter. Power spectrum analysis, revealing the power levels of different frequency components in the signal, was performed after applying a Hamming window function. Power was calculated in 1 -2 min time windows by integrating the root mean square value of the signal in frequency bands. Electroencephalographic seizures were defined as electroencephalogram patterns of high-amplitude rhythmic activity with evolution in frequency or amplitude that were at least three times higher than the baseline activities, lasted for at least 3-10 sec and abruptly reverted to baseline.

Animals that exhibited episode(s) of seizure-like EEG activity before the start of anesthesia were not included in the data analysis.

Carbetocin and oxytocin were administered by intracerebroventricular injection at the end of the surgery for the EEG electrode implantation. For intracerebroventricular injection animals were fixed in a stereotactic head holder. A 26 gauge needle attached to a Hamilton

microsyringe was lowered to a depth of 2.5 mm, and 1 .5 μΙ of saline solution containing 1 .5 pg of oxytocin or carbetocin was injected at a rate of 1 μΙ/min. The syringe was left in place for 1 .5 min before removal and wound closure. The EEG recording was started immediately upon completion of the surgery.

Example 3

In vitro electrophysiological recordings: Sprague-Dawley rats (P4-P6) were decapitated (P18- P22 rats were deeply anesthetized with isoflurane before decapitation) and the brains were dissected in cold (0°C-4°C) oxygenated (95% 0₂-5% C0₂) artificial cerebral spinal fluid containing: 120mM NaCI, 3 mM KCI, 25mM NaHC0₃, 1 mM NaH₂P0₄, 2.5mM CaCI₂, 2mM MgS0₄, 20mM glucose (300mOsm). Hippocampal coronal brain slices (400 pm) were cut with a vibrating-blade microtome Leica VT1000S (Leica Biosystems GmbH, Nussloch, Germany). The slices were maintained at room temperature for at least 1 hr in an interface holding chamber filled with humid 95% 0₂-5% C0₂ before transfer to a submersion experimental chamber superfused at 2 ml/min with artificial cerebral spinal fluid at 32°C-33°C.

Schaffer collaterals were stimulated at 0.05 Hz (100 μεβϋ, 20-40 μΑ) with a concentric bipolar stimulating electrode, and field excitatory postsynaptic potentials (fEPSPs) were recorded in the CA1 stratum radiatum using glass electrodes filled with 2M NaCI (3-6 ΜΩ resistance). Healthy slices were identified by the presence of a single population spike at a wide range of stimulus intensities and also by observing stable field potentials greater than mV requiring a similar amount of current for a period of 5 min prior to recording the experimental baseline. Slices not meeting these criteria were excluded.

Baseline fEPSPs were set to 40% of maximum response. A stable baseline was recorded for at least 15 min. Long-term potentiation (LTP) was induced using a high frequency stimulation (HFS) protocol (two 1 sec trains at 100Hz, separated by 20 sees). Data were collected using an Axopatch 1 D amplifier (Axon Instruments), filtered at 2 kHz and digitized at 5 kHz using a Digidata 1200 and Clampex 8 software (Axon Instruments). The slope of the fEPSP was measured after the end of the fiber volley in a 1 ms window. LTP was calculated as follows: LTP (%) = 100 x [mean slope of 10 fEPSPs 30 min after HFS / mean slope of 10 fEPSPs before HFS]. Paired pulse ratio of the fEPSP slope was obtained by stimulating 2 sequential fEPSPs per trace, 80ms apart (fEPSPI and fEPSP2) and calculated using the average of 10 traces with the following formula: Paired pulse ratio = fEPSP2 / fEPSPI . For experiments studying the effects of acute exposure to sevoflurane, the anesthetic was bubbled into the bath solution using a calibrated Penlon-Sigma vaporizer (Penlon, Abingdon, UK).

Example 4

Determination of activated cleaved caspase-3 using Western blot: Eighteen hours after exposure to sevoflurane, rat pups were deeply anesthetized with isoflurane and perfused with phosphate-buffered saline through the left cardiac ventricle. The brains were removed from the skull, put into liquid nitrogen and then stored at -80 °C until further use. On the day of the analysis the brain tissue was allowed to equilibrate to 4 °C. The tissue samples were then homogenized in 10% (w/v) RIPA buffer (50mM Tris pH 7.4, 150mM NaCI, 1 % NP-40, 0.5% sodium deoxycholate, 0.1 % SDS) containing complete protease inhibitors (Sigma, St. Louis, MO) and centrifuged at 16,000 g for 20 min. The supernatants were collected and quantified for protein concentration by bicinchoninic acid assay method (Bio-Rad Laboratories,

Mississauga, ON, Canada). Prepared protein samples (375 ug/well) were separated on 8-16% SDS-PAGE and transferred to a polyvinylidene fluoride membrane (Millipore, Billerica, MA). The membranes were blocked with 5% nonfat milk in 0.01 M phosphate-buffered saline with 0.1 % Tween-20 (pH 7.4) at room temperature for 1 h. Then, the membrane was incubated at 4 °C overnight with cleaved caspase-3 antibodies (Cell Signalling, Danvers, MA, 1 :500, diluted with 5% nonfat milk in 0.01 M phosphate-buffered saline with 0.1 % Tween-20). After three washes in 0.01 M phosphate-buffered saline with 0.1 % Tween-20, the membranes were incubated with horseradish peroxidase conjugated goat anti-rabbit serum (Santa Cruz

Biotechnology, Inc., Santa Cruz, CA) diluted at 1 :2000 in phosphate-buffered saline with 0.1 % Tween-20 for 1 h at room temperature. After three washes in 0.01 M phosphate-buffered saline with 0.1 % Tween-20 again, the blots were detected with Pierce ECL Western Blotting Substrate (Thermo Fisher Scientific, Rockford, IL). To prove equal loading, the blots were analyzed for γ- tubulin expression using an anti- γ-tubulin antibody (Santa Cruz). Exposure time was 10 mins for caspase-3, and 20 sees for γ-tubulin. The bands were semi-quantified with J-image software (NIH, Bethesda, MD). Western blot analysis for tissue samples from each animal was done in triplicate and reported as an average. Example 5

Statistical Analysis. Values are reported as mean ± SEM. SigmaStat 3.1 1 software (Systat Software, Inc, Point Richmond, CA) was used for statistical analysis. Single comparisons were tested using the t test, whereas multiple comparisons among groups were analyzed using ANOVA followed by Holm-Sidak tests. Changes over time in multiple groups were analyzed using repeated measures ANOVA followed by Holm-Sidak tests. Differences in proportions among groups were analyzed with a z-test. A P < 0.05 was considered significant.

Example 6

Seizure-like electroencephalographic activity in P4-P17 rats during sevoflurane anesthesia; effects of bumetanide: Arterial blood gas samples drawn at sevoflurane concentration of 2.1 % showed no evidence of either hypoxia or hypoventilation (p0₂: 429 ± 8; pC0₂: 46 ± 4; glucose: 1 18 ± 8); sevoflurane at 2.9%, on the other hand, caused respiratory depression in

spontaneously breathing rats. Because hypercarbia itself may increase seizure susceptibility (Yoshioka er a/., (1987) Pediatr. Neurol. 15: 36-40; Crawford er a/. , ( 1987) Can. J. Anaesth. 34: 437-4 ) and thus interfere with data interpretation of epileptogenic effects of sevoflurane, the concentration of sevoflurane of 2.1 % was used as the maintenance dose, while 6% sevoflurane for three min was used for anesthesia induction . Based on data from Orliaguet et al. (2001 ) Anesthesiology 95: 734-739), minimum alveolar concentration for sevoflurane in 9 day old and in 2 day old rats is 3.74% and 3.28%, respectively. Therefore, 2.1 % sevoflurane as used in this study was near 0.6 minimum alveolar concentration.

First, electroencephalographic activity was evaluated during 3 min of anesthesia induction with 6% sevoflurane and 30 min of anesthesia maintenance with 2.1 % sevoflurane in 39 rat pups that ranged in age from P4 to P17.

Start of induction of anesthesia with 6% sevoflurane was associated with a brief increase in electroencephalogram frequency followed by a gradual increase in amplitude and decrease in frequency that was followed by an abrupt and almost complete loss of electrical activity in older pups, as shown in Fig. 1 B. There was no such distinct transitional period during sevoflurane induction in younger rats.

A decrease in sevoflurane concentration from an induction dose of 6% to a maintenance dose of 2.1 % resulted in an increase in amplitude and frequency of the electroencephalogram in younger animals and caused burst suppression-like activity in some older rat pups (shown in Fig. 1 B). Only one rat (P5) of 39 P4-P17 rats studied exhibited a 58 sec episode of seizure-like electroencephalographic activity during 3 min of anesthesia induction with 6% sevoflurane.

During the first 30 min of anesthesia maintenance with 2.1 % sevoflurane, 8 of 20 P4-P8 rats (i.e. 40% of rats of this age category) exhibited multiple episodes of

electroencephalographic seizures that I combine lasted from 10 sec in one animal (P6) to 20.8 min in another (P5), with a mean seizure duration per animal of 86 ± 62 sec (n = 20). This seizure-like activity could be detected by recording both electroencephalogram 1 and electroencephalogram 2; most frequently it was present in electroencephalogram 2 only, though some animals exhibited seizure-like activity in electroencephalogram 1 but not in

electroencephalogram 2 (as shown in Fig. 1 C).

Within this age category, younger rats exhibited episodes of seizure-like

electroencephalographic activity more frequently than did their older counterparts. Thus, only 1 of 6 P7 rats had seizures, while seizures were observed in 6 of 12 P4-P6 rats. This trend in frequency of episodes of seizure-like activity in electroencephalogram continued in P10-P17 rats. These rat pups (n=19) did not exhibit any seizure-like EEG activity either during 3 min of 6% sevoflurane or 30 min of 2.1 % sevoflurane.

The possibility was tested that elevated concentrations of intracellular CI^", accumulated by NKCC1 , contribute to excitatory effects of sevoflurane in rats at earlier stages of brain development. The sulfamyl loop diuretic bumetanide at low concentrations is currently the only selective NKCC1 transporter inhibitor. The effect of bumetanide on frequency and duration of episodes of seizure-like activity during sevoflurane anesthesia was studied in P4-P9 rats. The P4-P9 rats were randomly distributed between two groups with similar number of animals of same age in both groups.

The animals in group one received bumetanide (5pmol/kg, intraperitoneally) 15 min prior to the anesthesia induction with 6% sevoflurane for 3 min, followed by anesthesia maintenance with 2.1 % sevoflurane for another 60 min. Rats in group two received the same volume of saline.

Seizures were observed in 12 of 20 animals that received bumetanide, compared to 12 of 9 rats that received saline. However, the duration of seizure activity per animal and number of episodes of seizures in the bumetanide-treated rats were significantly decreased, as shown in Figs. 2A-2C).

In some older rat pups (>P10) the emergence from 3 hours of anesthesia with 2.1 % sevoflurane resulted in more intensive seizures than those that were observed in P4-P8 rats during sevoflurane anesthesia. In contrast to seizures during anesthesia in younger rat pups, which were not readily associated with muscle movement, electroencephalographic seizures during emergence from anesthesia in older rat pups were accompanied by clonic/tonic muscle movements. Usually these emergence seizures occurred in several episodes starting between approximately 1 min and 15 min after termination of administration of sevoflurane (as shown in Figs. 3A-3C). These seizures lasted from 50 sec in one animal (P13) to 840 sec in another one (P14) with a mean duration of 486 ± 133 sec (n=6). Such emergence seizures were not prevented by pre-treatment with bumetanide administered 30 min prior to termination of anesthesia. To test the possibility that the observed emergence seizures are caused by low, sub-anesthetic doses of sevoflurane that may occur during terminal elimination of sevoflurane after the end of an anesthetic, the effects of 0.1 %, 0.2% and 0.5% sevoflurane applied for 60 min in P10-P17 rats were studied. These low doses of sevoflurane neither caused seizures nor any obvious increase in electroencephalographic activity in these animals.

Example 7

Bumetanide diminishes activation of caspase-3 in brain of neonatal rat pups exposed to sevoflurane or isoflurane: Whether sevoflurane anesthesia in neonatal rat pups causing neurotoxicity and whether this toxicity can be decreased by bumetanide was assessed.

Neonatal rats (P4) were exposed to sevoflurane as described above: 6% sevoflurane over 3 mins for induction and 2.1 % sevoflurane for 360 min for anesthesia maintenance. One half of these rats received bumetanide (5 pmol/kg, intraperitoneal^) 15 min prior to anesthesia with sevoflurane; the remainder received the same volume of saline. Rats in a control group were not exposed to sevoflurane. Again, absence of hypoxia and hypoventilation were assessed in a separate group of rat pups each anesthetized for 360 min and subjected to arterial blood gas sampling by cardiac puncture at the conclusion of the anesthetic. After emergence from anesthesia rats were tagged and returned to their dam. Eighteen hours after emerging from anesthesia animals were sacrificed and apoptotic changes in the brain were determined by evaluating activated caspase-3 using a Western blot technique.

Caspase activity was significantly increased in brain tissue of the sevoflurane anesthetized rats that received saline before exposure to sevoflurane. In contrast, animals that received bumetanide before exposure to sevoflurane had activated cleaved caspase-3 signals comparable to control rats, i.e. animals that were not exposed to sevoflurane anesthesia (as shown in Figs. 4A-4D).

Example 8

Anesthesia and electroencephalogram recording: To determine the effects of sevoflurane on cortical activity, rat pups ranging from postnatal day 4 to day 9 (P4-P9) were instrumented for electroencephalogram (EEG) recording, and off-line EEG analysis was performed as detailed previously (Edwards er a/. , (2010) Anesthesiology 1 12: 567-575). In brief, four electrodes of the headmounts of the EEG/EMG system (Pinnacle Technology, Lawrence, KS) were implanted under isoflurane anesthesia (1 .6-2.0%). No obvious differences in EEG activities were observed when the recordings were started either immediately or 1 to 2 days post surgery. Sevoflurane (Fushimi-machi, Osaka, Japan) anesthesia was induced with 6 % sevoflurane and 1 .5 L/min oxygen over 3 min, and maintained with 2.1 % sevoflurane and 1 .5 L/min oxygen over 30 min- 360 min in a thermostat chamber. Onset and offset of anesthesia were monitored via electroencephalogram and by loss and return of righting reflex, respectively. Anesthesia gas monitoring was performed using a calibrated Datex side stream analyzer that sampled from the interior of the animal chamber. To assure adequate oxygenation and respiration, some of the rat pups were anesthetized with the same sevoflurane protocol as rats in the study group. Arterial blood gas and glucose determinations were performed by cardiac puncture at the conclusion of the anesthetic. Arterial blood gases and glucose were measured using a portable clinical analyzer (i-STAT, Abbott Laboratories Inc., East Windsor, NJ). Electroencephalographic seizures were defined as electroencephalogram patterns of high-amplitude rhythmic activity with evolution in frequency or amplitude that was at least three times higher than the baseline activities, lasted for at least 3 s, and abruptly reverted to baseline.

Example 9

Measurements of acoustic startle response and prepulse inhibition of startle: The acoustic startle and prepulse inhibition (PPI) of startle tests were performed at P21 -P26 (most tests were performed at P24) using the SR-Lab startle apparatus (San Diego Instruments, San Diego, CA) as described in our recent paper (Cao et al. , Br. (2009) J. Pharmacol. 158: 2005- 2013). Rats were handled within 4 days prior to the test. Testing occurred during the light phase of the dark-light cycle. At the beginning of every testing session, each animal was placed into the cylindrical animal enclosure, and was then exposed to a 75 dB white noise (background) for a five-minute acclimation period. The acclimation period was then followed by a test session consisting of five different types of trials: 1 ) 120 dB pulse-only of 40 ms duration; 2-4) a 120 dB pulse of 40 ms duration preceded by a prepulse of 20 ms duration at 5 dB, 10 dB and 15 dB above background; and 5) a no stimulus trial of background noise. The delay between the onset of the prepulse and the onset of the pulse was 100 ms. The trials were presented in pseudorandom order, with variable inter-trial intervals, with an average duration of 15 s. The first four trials and last three trials consisted of 120 dB pulse- only trials. All five types of trials were presented eight times, each in pseudorandom order after the first four and last three pulse-only trials. The %PPI for each prepulse intensity was calculated using the following formula: %PPI=100 x [(pulse alone) - (prepulse +

pulse)]/pulse alone. The responses to the first four and last three pulse-only stimuli were not included in the calculations.

Example 10

Grooming behavior testing: The P21-P26 rats were video-recorded in a clear Plexiglas chamber (28 cm in diameter by 30.5 cm high). Each rat was placed individually in the chamber during the video recording. A camera lens was focused on the rat, providing a close-up view of the rat's face, forepaws, and upper body. Videotapes were played back at a speed of -4X for accurate scoring. The grooming behavior of each rat was analyzed during a period of 10 minutes. The four following behavioral elements, were analyzed:

Phase I, a series of elliptical strokes tightly around the nose; Phase 2, a series of unilateral strokes, each made by one paw that reaches from the vibrissae to below the eye; Phase 3, a series of bilateral strokes made by both paws simultaneously; the paws reach up past the ears and descend together over the front of the face; and Phase 4, a series of sustained bouts of body licking. The grooming behavior of the rat was considered as syntactic chain grooming if the rat completed a 4-phase series of events. More frequently, however, the rats exhibited out-of-order grooming chain sequences in which they would alternate between phase I and phase IV several times before resuming to phase I. This behavior was classified as nonsequential grooming. The data is presented as total time spent grooming, time spent in syntactic chain grooming and time spent in nonsequential grooming. The observer was unaware of treatment groups.

Example 11

Drugs: Oxytocin and carbetocin (15 μg/rat, i.e. v.) were administered as 1 .5 μΙ saline solution; aldosterone (20 mg/kg, s.c), and spironolactone (20 mg/kg, s.c.) were solubilized in DMSO at 12 mg/ml. Anti-TNF-a (25 μg/rat, i.p.) was diluted in saline and administered at a volume of 50 μΙ.

Example 12

Sevoflurane alters sensorimotor gating function and grooming behavior-effects responsive to pretreatment with bumetanide: Anesthesia of postnatal day 5-9 (P5-P9) rats with 2.1 % of sevoflurane (about 0.6 minimum alveolar concentration) does not cause hypoxia or hypoventilation, but is associated with episodes of cortical, encephalographically (EEG) detectable seizures. Also, six hours-long anesthesia of P4-P5 rats with sevoflurane caused an increase in levels of activated caspase-3. Both effects were diminished in animals that received bumetanide prior to start of sevoflurane.

To determine whether sevoflurane may also cause delayed behavioral defects and whether they are also responsive to pretreatment with bumetanide, prepulse inhibition (PPI) of acoustic startle response and grooming behavior in juvenile rats that were exposed to sevoflurane anesthesia during the early postnatal period were evaluated. The P4-P5 rats were anesthetized with 6% sevoflurane for 3 minutes for induction and 2.1 % sevoflurane for 357 min for anesthesia maintenance (6 hours in total). One half of these rats received bumetanide (5 μηΊθΙ/kg, i.p.) 15 min prior to anesthesia with sevoflurane; the remainder received the same volume of saline (Fig. 8A). Rats in a control group were not exposed to sevoflurane. Absence of hypoxia and hypoventilation were assessed in a separate group of rat pups each anesthetized for 360 min and subjected to arterial blood gas sampling by cardiac puncture at the conclusion of the anesthetic. After emergence from anesthesia, rats were tagged and returned to their dam. The PPI and grooming tests were performed when the rats achieved 21 -26 days of age. Most of the rats at the time of the tests were 24 days of age.

PPI of the startle response was significantly disrupted in animals that received saline before anesthesia with sevoflurane when compared to control animals

P<0.05; Fig. 8B). PPI was significantly impaired at prepulse intensities of 5 dB and 10 dB. Although, the decrease in %PPI in these rats was also greater at a prepulse intensity of 15 dB, it was not sufficient to reach a statistically significant difference. The bumetanide-treated group had PPI responses similar to controls (P=0.956 versus control and P=0.013 versus saline pretreated sevoflurane anesthetized group). Rats that received bumetanide (5 pmol/kg, i.p.) but were not exposed to sevoflurane anesthesia at P4, at P24 exhibited PPI of startle - not different from their counterparts that received equivalent volume of saline

P=0.92 ).

One-way ANOVA results established a significant difference among treatment groups in time spent in non-sequential grooming (Fig. 8C). This difference was due to the higher levels of grooming observed in rats that received saline as a pretreatment prior to anesthesia with sevoflurane ( F_(2i33)=5.421 , P<0.05 versus control and bumetanide groups; Fig. 8C). No significant differences were observed between the control rats and the animals pretreated with bumetanide prior to exposure to sevoflurane. Also, no significant differences were observed between all three treatment groups when time spent in syntactic chain grooming was compared

P=0.873). Because the saline-pretreated animals spent a little less time in syntactic chain grooming than rats in two other treatment groups, the increase in total time spent grooming by the saline-pretreated rats was not sufficient to achieve a statistically significant difference (F₍₂,₃3₎=2.883, P=0.07; Fig. 8C).

Example 13

Inhibition of aldosterone MRs diminishes impairment of PPI of startle and level of activated caspase-3, but not EEG seizures, caused by sevoflurane: Because general anesthesia is associated with increased aldosterone release and aldosterone efficiency can be higher in the neonatal brain (Geerling & Loewy (2009) Am. J. Physiol. Renal Physiol. 297: F559-576), the aldosterone receptor inhibitor, spironolactone, was used to test whether endogenous aldosterone may play a part in the adverse effects caused by anesthesia with sevoflurane in neonatal rats. To evaluate the effects of spironolactone on EEG activity, the P4-P9 rats were anesthetized with 6% sevoflurane for 3 minutes for induction and 2.1 % sevoflurane for 1 17 min for anesthesia maintenance (Fig. 9A). Spironolactone (20 mg/kg, s.c.) or equal volumes of DMSO were administered at 30 min after the start of sevoflurane.

Anesthesia of neonatal rats with sevoflurane was associated with episodes of cortical seizures. The animals that exhibited episode(s) of seizure-like activity before the start of anesthesia were not included in the data analysis. The administration of spironolactone was not associated with the decrease of EEG seizure activity previously observed in response to pretreatment with bumetanide. Seizures were observed in 4 of 6 animals that received spironolactone, with total duration of 60.0 ± 29.5 s and number of seizure episodes of 5.5±2.9 (Figs. 9B and 9C). Spironolactone markedly increased EEG seizures in 3 of 6 studied rats. The phenotype of this seizure activity was similar to one observed during sevoflurane anesthesia alone.

The exact anesthesia protocol that was previously used to determine the effect of bumetanide on changes in the level of activated caspase-3 caused by sevoflurane (Edwards ef a/. , (2010) Anesthesiology 1 12: 567-575) was repeated to test whether inhibition of aldosterone MRs may affect the level of activated caspase-3 increased by sevoflurane anesthesia. Instead of administering bumetanide 15 min prior to sevoflurane, spironolactone (20 mg/kg, s.c.) was injected (Fig. 10A). Other sevoflurane-anesthetized animals received an equal volume of DMSO. Rats in a control group were not exposed to sevoflurane, but received an injection of DMSO. The rats were sacrificed a day later, and the level of activated caspase-3 in brain tissue was determined. The DMSO-pretreated rats that were exposed to sevoflurane had significantly increased levels of activated caspase-3

(F(3,16₎=5.005, P<0.05 versus control), Fig. 10B). The spironolactone-pretreated rats that were exposed to sevoflurane, however, had levels of activated caspase-3 not different from control (P=0.867). The control animals that received spironolactone and were not exposed to sevoflurane tended to have levels of activated caspase-3 greater than the animals that received DMSO only or the spironolactone-sevoflurane group, but lower than those treated with DMSO and exposed to sevoflurane anesthesia. The increase in levels of activated caspase-3 in the spironolactone-only group was not sufficient to achieve a statistically significant difference.

To assess whether spironolactone may affect the sevoflurane anesthesia-induced abnormalities in PPI of startle, the P4 rats were subjected to 6 hrs of anesthesia as described above. Fifteen min prior to the start of anesthesia, rats were pretreated with either spironolactone (20 mg/kg, s.c.) or equal volume of DMSO (Fig. 1 1 A). PPI of startle in P24 rats that were pretreated with DMSO only prior to sevoflurane anesthesia at P4 was significantly disrupted

17.513, P<0.05 versus the spironolactone plus sevoflurane and spironolactone only groups), Fig. 1 1 B). Again, similar to the caspase-3 experiments, the level of PPI of startle exhibited by the spironolactone only group was between the values of PPI of startle of the sevoflurane only and the spironolactone plus sevoflurane groups. The grooming behavior of the same rats that were tested for PPI of startle response (above) was not different between all three treatment groups. The time spent grooming by the P24 rats that were pretreated with DMSO and then exposed to sevoflurane for 6 hrs was similar to time spent grooming by the rats that were pretreated with saline and then anesthetized with sevoflurane for the same duration (Fig. 1 1 C). The rats in the DMSO plus spirolactone group also spent a similar amount of time grooming, and this time tended to further increase in the spirolactone plus sevoflurane group. Example 14

Exogenous aldosterone exacerbates the sevoflurane-caused side effects: To test whether excess of aldosterone may further enhance the side effects caused by neonatal sevoflurane, the effects of exogenous aldosterone in the sevoflurane anesthetized rats were studied. First, we measured the effects of subcutaneously administered aldosterone on EEG activity of P4-P7 rats during anesthesia with sevoflurane. The baseline EEG was recorded for 60 min before start of administration of sevoflurane. The rats were anesthetized with 6% sevoflurane for 3 minutes for induction and 2.1 % sevoflurane for 1 17 min for anesthesia maintenance. Aldosterone (20 mg/kg, s.c.) was administered at 60 min after start of sevoflurane (Fig. 12A). Therefore, each animal served as its own control. The administration of aldosterone produced a marked increase in the number of episodes (t₍₆₎ = -2.739, P = 0.034) and total duration of EEG-detectable seizures (t₍₆₎ = -3.094, P = 0.021 ) (Figs. 12B and 12C). The duration of an individual episode of seizures also increased after administration of aldosterone; however, this increase was not sufficient to yield a statistically significant difference when compared to the prior aldosterone period. The subset of animals was treated with bumetanide (5 pmol/kg, i.p.) 60 min after administration of aldosterone, while anesthesia with 2.1 % sevoflurane was also maintained for another 60 min (total anesthesia duration in rats that received bumetanide lasted for 180 min). Bumetanide was not able to diminish aldosterone-enhanced seizures. In some animals, bumetanide further enhanced the intensity of seizures recorded after the administration of aldosterone (n=4). Importantly, the seizures after administration of aldosterone were more intensive than those seen in the presence of sevoflurane alone or after administration of spironolactone. Using the same experimental protocol, no seizure-like activity was observed during sevoflurane anesthesia either before or after administration of aldosterone and bumetanide in P17-20 rats (n=8) (Fig. 12D).

To test whether aldosterone affects the levels of activated caspase-3 in the sevoflurane-anesthetized animals, P4 rats were anesthetized with 6% sevoflurane for 3 min for induction and 2.1 % sevoflurane for 1 17 min for anesthesia maintenance (Fig. 13A). A shorter duration of anesthesia was chosen after observation of the intense seizures upon administration of aldosterone in the experiments described above. The rats received either aldosterone (20 mg/kg, s.c.) or equal volume of DMSO 15 min prior to anesthesia with sevoflurane. Rats in control groups were not exposed to sevoflurane. A subset of the control rats received aldosterone (20 mg/kg, s.c), and remaining control animals were administered an equal volume of DMSO (s.c). The animals were sacrificed 2 hrs after emergence from anesthesia, and level of activated caspase-3 was determined in the brain cortical tissue. The level of activated caspase-3 was significantly increased in the sevoflurane anesthetized rats that received DMSO before exposure to sevoflurane when compared to control rats that were not exposed to sevoflurane anesthesia (t₍i ₃) = -2.404, P = 0.032, Fig. 13B)).

Aldosterone did not increase caspase-3 activity in nonanesthetized animals but further increased it in the sevoflurane-anesthetized rats 1 .02, P<0.05).

The same treatment groups utilized for determination of caspase-3 activity were used to assess the effect of aldosterone on PPI of startle in the sevoflurane anesthetized animals. The rats were exposed to sevoflurane for 4 hours (Fig. 14A) instead of the 6 hour period that was employed in the experiments with sevoflurane anesthesia only (Fig. 1 A). Sevoflurane for four hours at P4 still resulted in significant impairment of PPI of startle measured at a prepulse intensity of 5 dB in the 24 day old rats (P<0.05 versus control) (Fig. 14B). The rats treated with aldosterone but not exposed to the anesthetic had a level of PPI not different from control (P=0.231 ). The animals pretreated with aldosterone and then anesthetized with sevoflurane exhibited significantly disrupted PPI at a prepulse intensity of 5 dB

P<0.05 versus all treatment groups). The impairment in PPI of startle in the sevoflurane anesthetized rats pretreated with aldosterone was significant at all three prepulse intensities.

The same rats that were tested for PPI of startle response were evaluated for their grooming behavior (Fig. 14C). The rats that received either DMSO or aldosterone but were not exposed to sevoflurane anesthesia exhibited similar time spent in grooming as the rats in control group reported in Fig. 14C. A four hour-long anesthesia with sevoflurane period slightly increased the time spent grooming by the DMSO-pretreated rats, and this time tended to further increase in the aldosterone plus sevoflurane group. However, no significant differences between all four treatment groups were observed

P=0.589).

Example 15

Oxytocin or its synthetic analog, carbetocin, alleviate side effects caused by sevoflurane anesthesia in neonatal rats: To assess the effects of oxytocin during anesthesia of neonatal rats with sevoflurane, the EEG activity, PPI of startle, and grooming behavior were studied. In the EEG experiments, a single dose of oxytocin or its synthetic analog, carbetocin, or equal volume of saline (control animals) were administered to P4-P6 rats by intracerebral injection at the end of the surgery for the EEG electrode implantation. A 26 gauge needle attached to a Hamilton microsyringe was lowered to a depth of 2.5 mm, and 1 .5 μΙ of saline solution containing 1.5 pg of oxytocin or carbetocin was injected at a rate of 1 μΙ/min. The syringe was left in place for 1 .5 min before removal and wound closure. The EEG recording was started immediately upon completion of the surgery. After baseline EEG recording for 15 min, anesthesia was induced with 6% sevoflurane for 3 min and maintained with 2.1 % sevoflurane for 57 min (Fig. 15A).

The rats pretreated with either oxytocin or carbetocin had significantly less EEG seizure activity when compared to the rats that received intracerebral administration of saline as a pretreatment ((t₍₆₎ = -2.739, P = 0.034) (Fig. 15B). All seizure parameters that were analyzed, such as duration of seizures (F_(2,17)=6.37, P<0.05), number of episodes of seizures

P<0.05), and duration of an individual seizure episode P<0.05), were diminished in the oxytocin- and carbetocin-pretreated rats when compared to the saline-pretreated animals. Intraperitoneal administration of carbetocin (20 mg/kg) to P5-P9 rats (n=10) did not result in depression of EEG seizures during sevoflurane anesthesia.

For the PPI of startle test, two groups of the P4 rats were pretreated either with carbetocin (1.5 g in 1.5 μΙ of saline, i.c.v.) or an equal volume of saline. Ten min after administration of carbetocin or saline, anesthesia was initiated with 6% sevoflurane and 2.1 % sevoflurane for 3 min and 117 min, respectively (Fig. 16A). The PPI of startle response, measured when the animals achieved 24 days of age, was significantly impaired in the saline-treated rats (t₍₇₎ = -2.9; P < 0.05 versus the carbetocin-treated animals, Fig. 16B. This difference was only detectable at a prepulse intensity of 5 dB. It has to be noted that PPI of startle was smaller in each treatment group across all three prepulse intensities when compared to the previous PPI tests. Both control and treatment groups of animals underwent surgical procedures for intracerebral injections.

The rats pretreated with carbetocin (1.5 pg, i.c.v.) prior to sevoflurane for 2 hrs spent significantly less time grooming (t₍₇₎=3.389, P<0.05 versus saline group, Fig. 16C). This difference was mostly due to a decrease in nonsequential grooming ((t₍₇₎=2.257, P=0.059 versus saline group), but it was not significant because of decrease in syntactic chain grooming, although it was much less (P=0.661 ).

Claims

CLAIMS We claim:

1. A method of reducing a side-effect of an anesthetic in an animal or human subject, the method comprising:

administering to an animal or human subject an effective dose of anesthetic; and administering to the animal or human subject at least one therapeutic agent selected from the group consisting of: a therapeutic agent characterized as decreasing the intracellular amount of CI^" in a recipient subject animal or human; a mineralcorticoid receptor (MR) antagonist, and an oxytocin receptor agonist;

wherein the therapeutic agent is administered before or with the anesthetic, thereby reducing the level of a side-effect of the anesthetic in the animal or human subject.

2. The method of claim 1 , wherein the at least one therapeutic agent characterized as decreasing the intracellular amount of CI^" in the recipient subject animal or human is an inhibitor of the Na⁺-K⁺-2CI^" symport ion co-transporter 1 (NKCC1 ).

3. The method of claim 1 , wherein the inhibitor of the Na⁺-K⁺-2CI^" symport ion co-transporter 1 (NKCC1 ) is selected from the group consisting of: furosemide, butmetanide (3-butylamino- 4-phenoxy-5-sulfamoyl-benzoic acid), ethacrynic acid, and benzmetanide, tripamide, or derivatives thereof.

4. The method of claim 3, wherein the inhibitor of the Na⁺-K⁺-2CI^" symport ion co-transporter 1 (NKCC1 ) is butmetanide.

5. The method of claim 4, wherein the inhibitor of the Na⁺-K⁺-2CI^" symport ion co-transporter 1 (NKCC1 ) is butmetanide and the anesthetic is sevoflurane.

6. The method of claim 1 , wherein the mineralcorticoid receptor (MR) antagonist is an aldosterone antagonist.

7. The method of claim 1 , wherein the mineralcorticoid receptor (MR) antagonist is spironolactone or a derivative thereof.

8. The method of claim 1 , wherein the mineralcorticoid receptor (MR) antagonist is 17- spironolactone.

9. The method of claim 1 , wherein the oxytocin receptor agonist is selected from the group consisting of: oxytocin , carbetocin, or an analog or a derivative thereof.

10. The method of claim 1 , wherein the anesthetic increases GABA_A/glycine receptor activity in the recipient subject animal or human.

1 1 . The method of claim 1 , wherein the anesthetic is isoflurane or sevoflurane.

12. The method of claim 1 , wherein the side-effect of the anesthetic is selected from the group consisting of: an induction of a seizure, a neurotoxicity, a behavioral effect, a cognitive effect, or any combination thereof.

13. A method of reducing a neural activity in an animal or human subject, the method comprising:

administering to an animal or human subject an effective dose of a mineralcorticoid receptor (MR) antagonist or an oxytocin receptor agonist;

wherein the mineralcorticoid receptor (MR) antagonist or oxytocin receptor agonist reduces the level of a neural activity in the animal or human subject.

14. The method of claim 13, wherein the neural activity is a seizure.

1 5. The method of claim 14, wherein the seizure is not a side-effect of an anesthetic.

16. The method of claim 1 3, wherein the mineralcorticoid receptor (MR) antagonist is spironolactone, or a derivative thereof.

17. The method of claim 13, wherein the oxytocin receptor agonist is selected from the group consisting of: oxytocin, carbetocin, or an analog or a derivative thereof.

18. A method of determining the likelihood of an animal or human subject developing a side- effect of an anesthetic administered to said subject, comprising determining whether said subject has an abnormality in a level of aldosterone or oxytocin compared to a normal level, or an abnormality in the physiological function or response thereto of aldosterone or oxytocin in the animal or human subject, wherein the presence of the abnormality indicates the likelihood of an animal or human subject developing a side-effect of an anesthetic administered to said subject.