WO2015081850A1 - Novel intravitreal injection drug delivery system for mouse nerve growth factor, and application thereof - Google Patents

Abstract

Disclosed is an intravitreal injection drug delivery system for mouse nerve growth factor (mNGF), the system being characterized by comprising the mNGF and an ultrasonic contrast agent. Also disclosed is an application of the intravitreal injection drug delivery system of the mNGF in the preparation of drugs for treating glaucomatous optic neuropathy, the drugs being suitable for intravitreal injection drug delivery under ultrasonic irradiation.

Description

Novel mouse nerve growth factor vitreous cavity injection drug delivery system and application thereof Technical field

The present invention relates to the field of medicaments for the treatment of ocular diseases by intravitreal injection of murine nerve growth factor.

Background technique

Nerve Growth Factor (NGF) is one of the earliest discovered neurotrophic factors. It is the most thorough research, and it has a neuronal growth regulator with neurotrophic and pro-apoptotic growth. It is central to the The development, differentiation, growth, regeneration and expression of functional properties of peripheral neurons have important regulatory effects. It can be used clinically to treat eye diseases such as optic nerve injury.

Mouse nerve growth factor (mNGF) is an intramuscular injection of NGF extracted from the submandibular gland of mice. It is a national class I new drug with a purity of over 98%. It has high homology with the structure of human NGF, and its biological effects are not obvious. Interspecific (human and mouse) specificity. It has been widely used clinically for the treatment of nerve damage recovery including optic nerve damage. We hope to be able to use these drugs for the treatment of fundus diseases, especially glaucoma. Corresponding to retinal diseases, only the application of murine nerve growth factor to the eye will achieve the desired therapeutic effect.

For the treatment of eye diseases, the current mode of administration of rat nerve growth factor is intramuscular injection, which requires systemic circulation to reach the eye tissue, which results in unsatisfactory therapeutic effects in the eye and also undergoes systemic metabolism. It has side effects on other tissues and organs. The reason may be that due to the particularity of the blood-eye barrier and the anatomy of the eye, the local effective concentration of the drug in the retina and optic nerve is low, resulting in poor efficacy, which makes its clinical application greatly restricted, so seek a A new type of drug delivery method that allows rat nerve growth factor to be more effectively and safely applied locally to the retina and optic nerve has become an urgent need for clinical treatment of eye diseases.

Since the presence of the blood-eye barrier makes the eyeball in a state of relative immune forgiveness, intraocular injection of exogenous antibodies can minimally trigger a potential immune response and inflammatory response, so the eyeball is an ideal organ for local treatment. The vitreous injection technique has the advantage of direct intraocular administration, which can avoid the side effects of other organs after systemic metabolism. However, at present, there have been no reports of intravitreal injection of mouse nerve growth factor for the treatment of eye diseases.

Summary of the invention

The present invention aims to provide a mouse intravitreal injection system for murine nerve growth factor, which has the problem of unsatisfactory therapeutic effect and large side effects in the administration mode of the existing mouse nerve growth factor for treating eye diseases mentioned in the background art. In order to break the existing mode of administration, the intravitreal injection of mouse nerve growth factor is used to treat eye diseases. This new administration method has the advantages of good therapeutic effect, safety and reliability.

In order to achieve the above object, the present invention provides such a vitreous nerve growth factor intravitreal injection administration system comprising: a mouse nerve growth factor and an ultrasound contrast agent.

Further, the ultrasonic contrast agent is sulfur hexafluoride microbubbles.

The present invention also provides the use of a murine nerve growth factor intravitreal injection system for the preparation of a medicament for the treatment of glaucomatous optic nerve damage, which is administered by intravitreal injection under ultrasound irradiation.

Further, the medicament is administered by intravitreal injection at an ultrasonic sound intensity of 0.5 W/cm ² and an irradiation time of 30 s to 60 s.

Further, the drug is injected at a dose of 12 ug/kg of mouse nerve growth factor.

Further, the drug is a stable suspension of murine nerve growth factor and an ultrasound contrast agent.

Further, the mouse nerve growth factor is mixed 1:1 with the ultrasound contrast agent in the suspension.

Beneficial effects:

Compared with the prior art, the present invention provides a novel ultrasonic microbubble-mediated local administration system for mouse nerve growth factor ophthalmology, which is administered by intravitreal injection, so that the route of the drug to the eye becomes simpler. Through systemic metabolism, the decomposition of the drug and the interference and destruction process of the drug in the body environment are avoided, thereby maximizing the concentration of the drug reaching the target organ, maximizing the therapeutic effect, and increasing the concentration of the drug in the retina. At the same time, it also reduces systemic toxic side effects, reduces the number of doses, delays or prevents the development of glaucomatous optic nerve damage, brings more effective treatment to glaucoma patients, reduces their blindness rate, and brings new complications to patients. The hope has greatly broadened the horizon of glaucoma optic nerve protection therapy and provided a new idea.

DRAWINGS

Figure 1 is a graph showing changes in intraocular pressure during different modeling periods.

detailed description

The present invention will be further described in detail below with reference to the accompanying drawings and specific embodiments. For the explanation of the present invention, the present invention is not limited to the following embodiments.

Intravitreal injection of mouse nerve growth factor:

Stable suspension of sulfur hexafluoride microbubbles and mouse nerve growth factor 1:1 dissolved in physiological saline

The above-mentioned drugs are administered by intravitreal injection:

Alccaine eye drops were anesthetized, and the compound tropicamide ocular drops were scattered. Under the operating microscope, a proper amount of aqueous humor was extracted from the transparent corneal area at the upper angle of the scleral margin. The upper limbus was 3 mm. The vertical sclera enters the needle and penetrates into the vitreous cavity. After the enlarged pupil is used to clear the tip of the needle in the vitreous cavity, the above drug is injected slowly. The rat nerve growth factor is injected at a dose of 12ug/kg. The cotton swab is pressed into the needle and the needle is taken out and given to the Taili. Eye drops must be taken to prevent infection. After closing the eyelid, the coupling agent is applied, and the ultrasonic probe is irradiated on the surface of the eyelid for ultrasonic irradiation, the ultrasonic sound intensity is 0.5 W/cm ² , and the irradiation time is 30 s-60 s.

The pharmacodynamic experiment of the mouse nerve growth factor intravitreal injection system for treating glaucomatous optic nerve damage provided by the invention:

In this study, we first constructed a model of high-eye optic nerve damage in rabbit eyes, and then combined the ultrasonic blasting microbubbles with mouse nerve growth factor to observe the therapeutic effect of ultrasonic blasting microbubbles on the damage of optic nerve in rabbits with high intraocular pressure. Analysis of the safety, feasibility and effectiveness of the drug delivery system provides a new idea for clinical treatment of glaucoma.

Experimental Materials

First, the main reagent

Second, the main equipment and instruments

Third, experimental animals

Healthy New Zealand white rabbits weigh 1.5kg, male or female, provided by Guangdong Medical Animal Experimental Center, with slit lamp, direct ophthalmoscopy, no obvious abnormalities in the anterior segment and fundus; Tono-pen tonometer for measuring intraocular pressure <21mmHg Animals can be used.

Experimental methods and steps

First, the experimental animal grouping

Twenty-five healthy New Zealand white rabbits were randomly divided into 5 groups, 5 (10 eyes each). Grouped as follows:

1, blank control group

2. High intraocular pressure model group

3, high intraocular pressure model + vitreous cavity injection mNGF group

4, high intraocular pressure model + vitreous cavity injection mNGF + ultrasound group

5, high intraocular pressure model + vitreous cavity injection mNGF + ultrasound + microbubble group

Second, the preparation of rabbit chronic high intraocular pressure model

Anesthetized animals were injected with 3% sodium pentobarbital (1 ml/kg) in the ear vein. After the surface anesthesia of ercaine eye drops, the sputum was opened and opened with a 1 ml syringe at 9 o'clock along the limbus. After withdrawing 0.2 ml of the anterior chamber water by 1 mm, 0.2 ml of a 0.3% compound carbomer solution was injected into the 1 mm anterior chamber of the contralateral limbus. The surgery will be eye-dropping. If intraocular pressure <22 mmHg was found during the experimental period, the injection was repeated 1 time as described above after 7 days. The model of optic nerve damage in rabbits with high intraocular pressure was controlled by intraocular pressure >22 mmHg and maintained for 4 weeks. The intraocular pressure at the same time point was measured daily using a Tono-pen pen tonometer and recorded.

Third, the preparation of the solution

(1) Preparation of compound carbomer solution

3.0 g of Carbomer-940 and 0.25 g of dexamethasone were weighed and dissolved in 1000 ml of physiological saline at a pH of 4.

(2) Preparation of mouse nerve growth factor solution for injection

The injected nerve growth factor was dissolved in sterile water for injection, and the rabbit was intravitreally injected with 18 ug/0.1 ml of a mouse nerve growth factor solution for injection.

(3) Preparation of microbubble suspension

Slowly inject sulphur hexafluoride microbubble lyophilized powder bottle with 0.9 ml of 0.9% physiological saline for standing. Use vigorous shaking before use to produce microbubbles with a microbubble concentration of 2×108/ml and an average diameter of microbubbles of 2.5 μm. 90% of the microbubbles have a diameter of <8 μm, an osmotic pressure of 290 Osm/kg, and isotonic with human plasma. The prepared microbubble solution is used up within 6 hours.

Fourth, the processing of each group

(1) blank control group

Intravitreal injection of PBS solution 0.1ml: ercaine eye drops topical anesthesia, compound tropicamide eye drops eye drops fully dilated, open sputum opening, 3% iodophor wash conjunctival sac, aseptic conditions Use a 1ml syringe to puncture the appropriate amount of aqueous humor in the transparent corneal area at the upper angle of the scleral margin, and then insert the needle into the vitreous cavity 3mm perpendicular to the sclera at the upper corner of the sclera. After the enlarged pupil, the tip of the needle is in the vitreous cavity. Slowly inject 0.1ml PBS solution, press the cotton swab into the needle, pull out the needle, observe the intraocular pressure and ocular vascular filling, give the ofloxacin eye drops, and dicoro eye ointment to prevent infection. The above operation was performed once a week for 3 weeks.

(2) High intraocular pressure model group

Do not deal with it.

(3) high intraocular pressure model + vitreous cavity injection mNGF group

Erkanin eye drops topical anesthesia, compound tropicamide eye drops, dilated eyes, open sputum opening, 3% iodophor wash the conjunctival sac, under sterile conditions with a 1ml syringe in the upper angle of the limbus At the transparent corneal area, a proper amount of aqueous humor was extracted from the tunnel, and then the needle was inserted into the vitreous cavity 3 mm perpendicular to the sclera at the upper corner of the sclera. After the dilated pupil was clear the tip of the needle in the vitreous cavity, 0.1 ml of mNGF solution was slowly injected, and the cotton swab was pressed. At the needle, pull out the needle, observe the intraocular pressure and ocular vascular filling, give the ofloxacin eye drops, and dicoro eye ointment to prevent infection. The above operation was performed once a week for 3 weeks.

(4) high intraocular pressure model + vitreous cavity injection mNGF + ultrasound group

The method of injecting mNGF into the vitreous cavity is the same as above. After the injection is completed, the rabbit eye is closed and the coupling agent is applied. The ultrasonic probe is placed on the eyeball at a frequency of 1 MHZ, and the sound intensity is 0.5 W/cm ^{2 to} illuminate the eyeball for 60 s. The above operation is performed once a week for 3 times. week.

(5) high intraocular pressure model + vitreous cavity injection mNGF + ultrasound + microbubble group

Rabbit dilated and anesthetized in the same way as before, use a 1ml syringe to penetrate the vitreous cavity 3mm away from the corneal cavity to aspirate the corresponding amount of vitreous humor, then inject 0.1ml mNGF solution, the specific operation is the same as above; then inject 0.1ml microbubbles from the same site The suspension was incubated with the coupling agent after the rabbit eyes were closed, and the above parameters were subjected to ultrasonic irradiation once a week for 3 weeks.

Experimental result

First, intraocular pressure measurement

As shown in Table 1, the intraocular pressure of the model group before the modeling was 15.0±2.0 mmHg, and the intraocular pressure of the control group was 13.6±1.5 mmHg. There was no significant difference in the intraocular pressure between the two groups (P=0.137); The intraocular pressure of the model group at 1 week, 2 weeks, and 4 weeks after the model was established (33.4±2.8, 34.1±2.5, and 34.8±2.2 mmHg), and the intraocular pressure of the control group (13.6±1.8, 13.4±1.7). , 13.3 ± 1.4 mmHg) was significantly statistically significant (P = 0.000). As can be seen from Figure 1, the increase in intraocular pressure caused by the model can be maintained above 30 mmHg for 4 weeks.

Table 1 Comparison of intraocular pressure between control eye and model eye

Note: Independent sample t test

Second, rabbit living eye examination

1, the rabbit eye anterior photography

Blank control group: the rabbit cornea is transparent, the anterior chamber depth is normal, the aqueous humor is clear, and the iris texture is normal. The pupil is 3 × 3 mm and the lens is transparent.

High intraocular pressure model group: rabbit conjunctival mixed hyperemia, conjunctival vascular tortuosity, central corneal fog edema, anterior chamber deepening, aqueous humor is basically clear, iris texture is unclear, pupil dilated, the lens is still transparent.

High intraocular pressure model + vitreous cavity injection mNGF group or + ultrasound group and high intraocular pressure model + vitreous cavity injection mNGF + ultrasound + microbubble group: rabbit anterior segment changes were consistent with the high intraocular pressure model group.

2, morphological observation of rabbit fundus

The blank control group: the retina of the rabbit's fundus was orange-red, the optic disc was elliptical, the boundary was clear, and the color was reddish; the cup was irregularly petal-shaped, and the disc edge was wider; a pair of accompanying retinal central motions were emitted from both sides of the optic disc. , vein, horizontal trend, arteriovenous ratio 1:2, peripheral blood vessels are radial.

High intraocular pressure model group: faintly visible retina, pale, rabbit retina atrophy, a large number of granular and flaky pigmentation; optic disc edema, border is not clear, the color is light; the cup is obviously enlarged, the color is pale, the disc area is obvious Narrowed; the retinal blood vessels become thinner and climb out of the edge of the optic disc.

High intraocular pressure model + vitreous cavity injection mNGF group or + ultrasound group: compared with the high intraocular pressure model group, the rabbit retina has a reddish color and mild retinal atrophy; the optic disc boundary is unclear, mild edema, and the color becomes reddish; The cup became smaller and the area along the disc was significantly wider; the retinal blood vessels were not significantly thinned.

High intraocular pressure model + vitreous cavity injection mNGF + ultrasound + microbubble group: rabbit retina reddish color, clear disc border, color reddish; visual cup higher intraocular pressure model group significantly smaller, disc area significantly widened; retinal blood vessels The thickness and direction are basically normal.

3, rabbit F-VEP detection

The comparison results of the F-VEP latency and amplitude of each group are shown in Table 2.

After one-way analysis of variance, the differences of N1, P1 and N2 latency in each group were statistically significant (P=0.000). After pairwise analysis, the incubation periods of N1, P1 and N2 in the high intraocular pressure model group were significantly higher than those in the blank control group. The difference was statistically significant (P<0.05); the high intraocular pressure model + vitreous injection group or the ultrasound group N1, P1, N2 latency group was significantly shorter, the difference was statistically significant ( P<0.05); high intraocular pressure model + vitreous cavity injection mNGF + ultrasound + microbubble group N1, P1, N2 latency higher intraocular pressure model + vitreous cavity injection mNGF group or + ultrasound group shortened, the difference was also statistically significant (P <0.05). The difference of amplitude of N1-P1 in each group was statistically significant (P=0.000). After pairwise comparison analysis, the amplitude of N1-P1 in the high intraocular pressure model group was significantly lower than that in the blank control group. Low, the difference was statistically significant (P<0.05); high intraocular pressure model + vitreous injection mNGF group or + ultrasound group N1-P1 amplitude higher intraocular pressure model group significantly increased, the difference was statistically significant (P <0.05); high intraocular pressure model + vitreous cavity injection mNGF + ultrasound + microbubble group N1-P1 amplitude higher intraocular pressure model + intravitreal injection of mNGF group or + ultrasound group, the difference was also statistically significant (P<0.05 ).

Table 2 Comparison of latency (ms) and amplitude (nV) of F-VEP in each group

Note: One-way analysis of variance, △ ※ indicates that there was no statistically significant difference between the two groups (P>0.05), there was significant statistical difference between the two groups (P<0.05).

4, Cirrus OCT measurement of rabbit retinal thickness comparison

The average thickness of the retina of 1-5 rabbits measured by Cirrus OCT was 226.20±4.49, 194.00±5.40, 206.50±2.10, 207.15±2.40 and 211.50±3.41μm, respectively.

After one-way analysis of variance, the mean thickness of retinal of each group was statistically significant (P=0.000). After pairwise analysis, the average thickness of retina in rabbits with high intraocular pressure was significantly thinner than that of blank control group. The significance of the study (P<0.05); the high intraocular pressure model + vitreous cavity injection mNGF group and high intraocular pressure model + vitreous cavity injection mNGF + ultrasound group rabbit retina average thickness was significantly thicker than the high intraocular pressure model group, the difference was statistically significant ( P<0.05), but there was no statistically significant difference between the 3 and 4 groups (P>0.05). The average thickness of the retina in the high intraocular pressure model+vitreous injection mNGF+ultrasound+microbubble group was significantly thicker than that in the high intraocular pressure model group. It was still thinner than the blank control group, and the difference was statistically significant (P<0.05).

Third, rabbit retina and optic nerve histopathological examination

3.1 Morphological observation of rabbit retina

The blank control group: the layers of the rabbit retina are clear, arranged closely and neatly; the photoreceptor cells are arranged neatly in a regular brush shape; the outer nuclear layer has the largest number of cells, the nuclei are small, the staining is deep, and the arrangement is dense; Neat, large nuclei, slightly darker; inner plexiform layer and outer plexiform layer have obvious reticular structure; ganglion cell layer is arranged in a single layer, and the nucleus is large and stained lightly, round or oval. The cell boundaries are clear.

High intraocular pressure model group: the structure of the retina is disordered, loose, and the level is unclear; the photoreceptor cell layer is disorderly arranged; the outer nuclear layer cells are disorderly arranged and the thickness is thin; the inner layer cells are loosely arranged and disordered, and a large number of vacuolar cells are visible. The inner plexiform layer and the outer plexiform layer are disordered, slightly thinned, and the ganglion cell layer is unclear; the number of ganglion cell layer cells is significantly reduced, showing vacuolization.

High intraocular pressure model + vitreous cavity injection mNGF group or + ultrasound group: the structure of the retina is relatively complete, the stratification is relatively clear; the photoreceptor cell layer is loosely arranged; the outer nuclear layer cells are arranged irregularly; the inner nuclear layer cells are slightly loose. It can be seen that the vacuolar cells are scattered; the thickness of the plexiform layer and the outer plexiform layer is thinner; the number of cells in the ganglion cell layer is higher than that in the intraocular pressure model group, and the cells scattered in the vacuole are still visible.

High intraocular pressure model + vitreous cavity injection mNGF + ultrasound + microbubble group: the structure of the retina is relatively complete, the layering is clear; the junction of the inner and outer nodes of the photoreceptor cell layer is clear and neatly arranged; the inner and outer nuclear layer cells are arranged regularly. The thickness is normal; the thickness of the plexiform layer and the outer plexiform layer is basically normal; the number of cells in the ganglion cell layer is obviously increased, the morphology is generally normal, and the vacuolar-like cells are even visible.

3.2 Light microscopy measurement of rabbit retinal thickness and retinal RGCs count

3.2.1 Light microscopy measurement of rabbit retina thickness

The average values of the retinal thickness of 1-5 rabbits were 289.30±2.39, 239.15±2.68, 254.50±3.03, 255.05±2.28 and 269.50±3.00μm, respectively.

After one-way analysis of variance, the difference of retinal thickness in each group was statistically significant (P=0.000). After pairwise analysis, the retinal thickness of rabbits in the high intraocular pressure model group was significantly thinner than that of the blank control group. Learning significance (P<0.05); high intraocular pressure model + vitreous injection mNGF group or + ultrasound group rabbit retina thickness was significantly thicker than high intraocular pressure model group, the difference was statistically significant (P<0.05), but groups 3 and 4 There was no statistically significant difference (P>0.05). The high intraocular pressure model+vitreous injection of mNGF+ultrasound+microbubble group was significantly thicker than the high intraocular pressure model+vitreous injection of mNGF or +ult ultrasound group. However, it was still thinner than the blank control group, and the difference was statistically significant (P<0.05).

3.2.2 Light microscopy measurement of RGCs counts in rabbit retina

In the toluidine blue staining of retinal smear, the RGCs were larger, the cytoplasm was filled with Nissl bodies, the nucleus was lightly stained, and the nucleolus was obvious; while the amacrine cells were relatively small, mostly round or oval. Shape, less cytoplasm, darker cell staining. Therefore, the two cells are roughly distinguished by the size and shape of the cells and the depth of nuclear staining.

The RGCs counts of rabbit retinas in groups 1-5 were 26.04±0.70, 14.97±1.30, 19.33±0.78, 20.25±0.98 and 23.97±0.90, respectively.

After single factor analysis of variance, the difference of RGCs counts in each group was statistically significant (P=0.002). After pairwise analysis, the RGCs counts in the high intraocular pressure model group were significantly lower than those in the blank control group, and the difference was statistically significant ( P<0.05); the high intraocular pressure model + intravitreal injection of mNGF group or + ultrasound group RGCs count was significantly higher than the high intraocular pressure model group, the difference was statistically significant (P <0.05), but there was no significant statistical comparison between groups 3 and 4. Learning differences (P>0.05); high intraocular pressure model + vitreous cavity injection mNGF + ultrasound + microbubble group RGCs count was significantly higher than high intraocular pressure model + vitreous injection mNGF or + ultrasound group, but still lower than the blank control group, the difference Statistically significant (P < 0.05).

3.3 Quantitative analysis of rabbit optic axon light microscopy

In the Nissl staining of rabbit optic nerve, the nucleus is light blue, and the neuron cells are rich in Nissl. It is a basic substance in the cell, dark blue, plaque or granular. In the blank control group, more N-nose and dark optic axons stained deep blue were observed under light microscopy.

The number of axons of rabbits in groups 1-5 is shown in Table 3.

After one-way analysis of variance, the number of optic axons, the diameter of optic nerve axons, and the percentage of axons in the optic nerve area of each group were statistically significant (P=0.000, 0.001 and 0.02); In the model group, the axons of the axons were swollen, the number was decreased, the diameter of the optic nerve axons was increased, and the percentage of optic nerve axons in the cross-sectional area of the optic nerve was decreased, which was significantly different from the blank control group (P<0.05). After the intravitreal injection of mNGF, The number of optic nerve axons increased, the diameter of optic nerve axons decreased, and the percentage of optic nerve axons in the cross-sectional area of the optic nerve increased, which was significantly different from the high intraocular pressure model group (P<0.05). In the application of vesicles, the degree of loss of myelin sheath in the optic nerve is significantly reduced, the swelling of the optic nerve axon is reduced, and the number is increased. Compared with the high intraocular pressure model group, the high intraocular pressure model + the vitreous cavity injection mNGF group or the + ultrasound group, it is significant. Statistical difference (P < 0.05).

Table 3 Quantitative image analysis results of rabbit optic axons

Note: One-way analysis of variance, △ ※ indicates that there was no statistically significant difference between the two groups (P>0.05), there was significant statistical difference between the two groups (P<0.05).

4. Ultrastructure of rabbit retina and optic nerve observed by transmission electron microscopy

4.1 Ultrastructural observation of rabbit retina

The blank control group: photoreceptor cells (rods, cones) have a clear structure and neatly arranged; the nuclear regions of the photoreceptor cells constitute the outer nuclear layer of the retina, arranged neatly, with uniform nuclear chromatin distribution, containing more mitochondria and other organelles; The nuclear region of the polar cell constitutes the inner nuclear layer, arranged neatly, and the nuclear chromatin is uniform; the ganglion cells are round or oval, the nuclear membrane is clear, the chromatin is evenly distributed, the nucleolus is obvious, the organelle mitochondria, the rough endoplasmic reticulum, the Golgi apparatus Rich.

High intraocular pressure model group: photoreceptor cells partially ruptured, outer membrane disc outline is blurred, mitochondria are swollen to varying degrees, vacuolar degeneration; outer nuclear layer cells are disordered, loose, nuclear chromatin is uneven, mitochondria are obviously swollen and vacuolar-like The number of ganglion cells is reduced, swelling, pale, microfilaments, microtubule components are reduced, and organelles such as mitochondria, endoplasmic reticulum and Golgi are basically disappeared.

High intraocular pressure model + vitreous cavity injection mNGF group or + ultrasound group: the photoreceptor extracellular membrane disc structure is still visible, the arrangement is light and disordered; the nuclear membrane of the outer nuclear layer is basically intact, the nuclear chromatin is relatively uniform, and no obvious vacuolar changes are observed in the mitochondria. The nucleus layer cells are slightly swollen, the chromatin edge set; the ganglion cell layer envelope is clear, the nucleus is filled Fully uniform euchromatin, mild mitochondrial vacuolar degeneration.

High intraocular pressure model + vitreous cavity injection mNGF + ultrasound + microbubble group: photoreceptor cells are arranged neatly, mitochondria showed no obvious swelling and vacuolization; outer nuclear nucleus chromatin uniform; nuclear layer nuclear membrane is not clear, mitochondrial changes are not obvious Most ganglion cells have clear nuclear membrane, uniform chromatin distribution, obvious nucleoli, clear mitochondria, and rough endoplasmic reticulum.

4.2 Observation of ultrastructure of rabbit optic nerve

The blank control group: the optic nerve axon rule, the myelin sheath structure is intact; clear microtubules, microfilaments and mitochondria and other organelles can be seen in the axoplasma.

High intraocular pressure model group: optic nerve myelin dissolution, loose; axon structure disorder, microtubule and microfilament structure disappeared, mitochondria swelling, vacuolar degeneration.

High intraocular pressure model + vitreous cavity injection mNGF group: optic nerve axons are irregular in size, disordered in arrangement, partial myelin sheath is thin, loose, and separated; microtubules and microfilaments in axons are swollen, but do not disappear, mitochondrial partial vacuolar degeneration .

High intraocular pressure model + vitreous cavity injection mNGF + ultrasound group: the myelin sheath of the optic nerve axon is thin, disordered and loose; microtubules and microfilaments are visible in the axons, and some mitochondrial vacuolar degeneration.

High intraocular pressure model + vitreous cavity injection mNGF + ultrasound + microbubble group: optic nerve myelin structure is intact, densely arranged, but not neat; microtubules and microfilaments in axons are clear, no obvious mitochondrial vacuolar degeneration.

5. Immunohistochemical staining for the determination of protein expression of apoptosis-related factors Bcl-2 and Bax in cells

In the blank control group, the expression of Bcl-2 and Bax was not obvious in brown retina. In the high intraocular pressure model group, more brown-yellow Bax expression was observed in the retinal inner nuclear layer and ganglion cell layer, while Bcl- 2 expression decreased; high intraocular pressure model + vitreous injection of mNGF or + ultrasound group rabbit retinal inner nuclear layer and ganglion cell layer showed less brown-yellow Bax expression, while Bcl-2 expression increased; high intraocular pressure model + Intravitreal injection of mNGF + ultrasound + microbubble group of rabbit retina can be seen in the brown-yellow Bax expression, and the expression of Bcl-2 increased significantly.

After one-way analysis of variance, it can be seen from Table 4 that the expression of Bcl-2 was decreased, the expression of proapoptotic gene Bax was increased, and the ratio of Bcl-2/Bax was decreased in the high intraocular pressure model group compared with the blank control group. There was a statistically significant difference in the expression of Bcl-2, Bax and Bcl-2/Bax gene between the two groups (P<0.05), indicating that the proapoptotic protein performs apoptosis program in the high intraocular pressure model. It tends to be apoptotic. along with The application of mNGF, the expression of Bcl-2 gene protein began to increase, the expression of Bax gene protein began to decrease, the ratio of Bcl-2/Bax increased, and the gene proteins of Bcl-2, Bax and Bcl-2/Bax between each group There was no statistically significant difference in expression (P>0.05). However, compared with the high intraocular pressure model group, the expression of Bcl-2, Bax and Bcl-2/Bax gene protein was statistically significant (P<0.05). mNGF exerts neuroprotective effects, and the inhibitory protein performs inhibition of apoptosis, and cells tend to survive. However, ultrasound irradiation alone after intravitreal injection of mNGF did not significantly reduce apoptosis. With the application of mNGF combined with ultrasound microbubbles, the expression of anti-apoptotic gene Bcl-2 was significantly increased, the expression of proapoptotic gene Bax was significantly decreased, and the ratio of Bcl-2/Bax was increased. Compared with other groups, Bcl-2 There was a statistically significant difference in the expression of Bax and Bcl-2/Bax gene proteins (P<0.05), which indicated that mNGF was significantly enhanced by ultrasound microbubbles and the cell survival was significantly increased.

Table 4 Immunohistochemical staining and RT-PCR method for detecting protein expression and mRNA expression of Bcl-2 and Bax in rabbit retina

Note: One-way analysis of variance, △ ※ indicates that there was no statistically significant difference between the two groups (P>0.05), there was significant statistical difference between the two groups (P<0.05).

Detection of mRNA expression of Bcl-2 and Bax in cells by real-time PCR

As can be seen from Table 4, by single factor analysis of variance, the expression of Bcl-2 mRNA in the high intraocular pressure model group decreased, and the mRNA expression of the proapoptotic gene Bax increased. Compared with the blank control group, the two groups There was a statistically significant difference in the expression of Bcl-2 and Bax mRNA (P<0.05), indicating that the expression of Bcl-2 and Bax was changed under high intraocular pressure, and the proapoptotic gene was involved in the apoptosis program. The cells tend to apopulate. The expression of Bcl-2 mRNA in the high intraocular pressure model + vitreous cavity injection mNGF or + ultrasound group was increased, and the mRNA expression of the proapoptotic gene Bax was decreased. Compared with the high intraocular pressure model group, Bcl-2 There was a statistically significant difference between the mRNA expression of Bax and Bax (P<0.05), which indicated that mNGF played a role, and the expression levels of Bcl-2 and Bax changed, which led to a decrease in apoptotic cells and an increase in viable cells. The expression of Bcl-2 mRNA in the high intraocular pressure model + vitreous cavity injection mNGF+ultrasound+microbubble group was further increased, and the mRNA expression of the proapoptotic gene Bax was further decreased. Compared with other groups, Bcl-2 There was a statistically significant difference between the mRNA expression of Bax and Bax (P<0.05).

VII. Detection of NO, MDA and SOD in rabbit retina

It can be seen from Table 5 that the NO content of the retina in the blank control group was 23.384±3.282umol/g and the MDA content was 7.415±0.3444nmol/mg prot. The content of NO and MDA in the retinal tissue of each group increased. High, there was a statistically significant difference between the groups (P = 0.000). After two-two comparison analysis, the contents of NO and MDA in the retina of the high intraocular pressure model group were significantly increased, which was significantly different from the blank control group (P<0.05). There was no significant difference in the content of NO and MDA between the high intraocular pressure model + vitreous cavity injection mNGF group or + ultrasound group, high intraocular pressure model + vitreous cavity injection mNGF + ultrasound + microbubble group (P>0.05). ), but there was a statistically significant difference (P<0.05) compared with the blank control group and the high intraocular pressure model group.

After one-way analysis of variance, the activity of SOD in the blank control group was 75.147±2.375U/mgprot, and the activity of SOD in the retina of each group was significantly decreased. There was significant difference between the groups (P=0.000). After two-two comparison analysis, the activity of SOD in the retina of rabbits with high intraocular pressure was significantly decreased, and there was significant statistical difference between the two groups (P<0.05). High intraocular pressure model + vitreous cavity injection mNGF group or + ultrasound group, high intraocular pressure model + vitreous cavity injection mNGF + ultrasound + microbubble group rabbit retina SOD activity increased in the intraocular pressure model group, but groups 3, 4, 5 There was no significant difference between the two comparisons (P>0.05).

Table 5 Changes in NO, MDA and SOD contents in rabbit retina

Note: One-way analysis of variance, △ ※ indicates that there was no statistically significant difference between the two groups (P>0.05), there was significant statistical difference between the two groups (P<0.05).

discuss

1. Preparation of rabbit high-eye optic nerve damage model

Glaucoma animal models are very important in the study of glaucoma. At present, many studies have proposed a variety of glaucoma animal models, the most ideal of which is laser ablation of trabecular meshwork to induce monkey high intraocular pressure model, but its price is expensive, the source is scarce, and it is impossible to conduct experiments in large quantities, which limits the application of research. The early attempts of the researchers to make a glaucoma model began with the production of a rabbit eye glaucoma model. The rabbit eyes were large, easy to operate, and low in cost, but the intraocular inflammation was also severe. Rabbit eye glaucoma model generally chooses anterior chamber injection of chymotrypsin, methyl cellulose, compound carbomer, etc. In this experiment, rabbit anterior chamber injection compound carbomer was selected for modeling. The carbomer solution is gelatinous at a pH of 6 to 12, and the pH of the aqueous humor is about 7. After it is injected into the anterior chamber, the carbomer changes from a solution state to a gel, which not only blocks the corner. And the trabecular meshwork prevents the discharge of aqueous humor, quickly becomes gelatinous and is not easy to flow back from the injection point ^[12] . During the experiment, the average intraocular pressure of the high intraocular pressure model group was 32-37 mmHg, and the high intraocular pressure lasted for more than 4 weeks. This is consistent with previous studies. The injection of the compound carbomer solution in the anterior chamber is an ideal high. Intraocular pressure modeling method.

The visual evoked potential is an electrical response to the visual stimuli in the occipital region of the cerebral cortex. It is a sensitive means of evaluating the nerve damage caused by the retina receiving stimulation and conduction through the visual pathway to the occipital cortex. The lesion between the retina and the visual cortex, the latency and amplitude of F-VEP mainly reflect the functional state of the optic nerve myelin and axon. Therefore, for this experiment, F-VEP is a better means to evaluate the function of the optic nerve. The results of this experiment showed that the N1 latency, P1 latency and N2 latency of F-VEP in the high intraocular pressure membrane group were significantly longer than those in the blank control group, and the amplitude of N1-P1 wave was significantly decreased, which was statistically significant.

The results of OCT and light microscopy of rabbit retina thickness showed that the retinal thickness of rabbits in the high intraocular pressure model group was significantly thinner, indicating that chronic high intraocular pressure caused certain damage to the retina of the rabbit, which made the thickness of the retina thinner overall. In this study, the morphology of the retina of rabbit retina was found to be disordered, loose, and unclear in the layers of the high intraocular pressure model group; the photoreceptor cell layer was disordered; the outer nuclear layer cells were disordered and the thickness was thin; Loose, disordered, a large number of vacuolar cells can be seen; the inner plexiform layer and the outer plexiform layer are disordered, slightly thinned, and the ganglion cell layer is unclear; the number of ganglion cell layer cells is significantly reduced, showing vacuoles Sample change. This indicates that the tissue structure of the retina is damaged under the action of chronic high intraocular pressure. The rabbit retinal RGCs count high intraocular pressure model group and the blank control group were statistically significant, indicating that chronic high intraocular pressure mainly acts on the ganglion cell layer, causing apoptosis and the number of ganglion cells to decrease. In addition, rabbit optic axons may be damaged after continuous high intraocular pressure, the axons of the optic nerves are swollen, the number is decreased, the number of optic nerve axons in the high-eye membranous group is reduced, the diameter of the optic nerve axons is increased, and the axons of the optic nerve occupy the cross-sectional area of the optic nerve. The percentage was reduced and there was a statistically significant difference compared to the blank control group.

In this study, the ultrastructure of rabbit retina and optic nerve under high intraocular pressure was observed by transmission electron microscopy. It was found that the photoreceptor cell membrane profile of the high intraocular pressure model group was blurred, the mitochondria were swollen to varying degrees, and the vacuolar degeneration; the outer nuclear layer cells were disordered and loose. The nuclear chromatin is uneven, the mitochondria are obviously swollen and vacuolized. The number of ganglion cells is reduced, the swelling and color are light, the microfilament and microtubule components are reduced, and the organelles such as mitochondria, endoplasmic reticulum and Golgi are basically disappeared. The axons of the optic nerve are disordered, the myelin is dissolved, loose, the microtubules and microfilaments disappear, the mitochondria are swollen, and the vacuoles are degenerated.

In summary, this experiment confirmed the preparation of rabbit high-ocular optic nerve damage animal model from the aspects of structure and function, pathological histomorphology and ultrastructure, which laid the foundation for the next experiment.

2. Ultrasound microbubble mediates the protective effect of optic nerve growth factor on optic nerve damage in rabbits with high intraocular pressure

We performed high-intensity rabbits with successful modeling, and observed the structure and function of rabbit retina and optic nerve, macroscopic and ultrastructural methods, and explored the effect of ultrasound microbubble-mediated optic nerve growth factor on high intraocular pressure rabbits. The protective effect of optic nerve damage. The results of this experiment showed that the N1, P1, and N2 latency of F-VEP was shorter in the intraocular pressure group than in the intravitreal injection of mouse nerve growth factor. The amplitude of N1-P1 wave was higher in the ocular membranous group, which was more significant. Statistical difference (P<0.05), indicating that intravitreal injection of mouse nerve growth factor has a protective effect on rabbit optic nerve damage. After intravitreal injection of mouse nerve growth factor, ultrasound irradiation alone showed that there was no statistically significant difference in the N1, P1, N2 latency and N1-P1 wave amplitude of F-VEP compared with pure intravitreal injection of mouse nerve growth factor (P> 0.05), This indicates that the simple irradiation of ultrasound does not increase the protective effect of rat nerve growth factor. This is because no microbubbles are given by ultrasound irradiation alone, no cavitation effect is produced, and no drug that increases the concentration of optic nerve growth factor to the retina is obtained. concentration. Ultrasound microbubbles combined with intravitreal injection of mouse nerve growth factor, F-VEP N1, P1, N2 latency was significantly shortened, N1-P1 wave amplitude was significantly increased, compared with ultrasound irradiation directly after intravitreal injection of mouse nerve growth factor There was a statistically significant difference (P<0.05), which indicated that the rat nerve growth factor was more effective in promoting the functional recovery of optic nerve damage caused by high intraocular pressure under the action of ultrasonic blasting microbubbles.

In this study, the thickness of rabbit retina was measured by light histopathology and OCT. The intraocular pressure of the intraocular lens was significantly thicker in the intraocular pressure group. The difference was statistically significant (P<0.05). This indicates that mouse nerve growth factor has a protective effect on high intraocular pressure optic nerve damage. Ultrasound microbubbles combined with intravitreal injection of murine nerve growth factor in light microscopic retinal thickness measurements were significantly different from those obtained by intravitreal injection of mouse nerve growth factor or intravitreal injection of mouse nerve growth factor alone. <0.05), which also indicates that ultrasonic blasting microbubbles can significantly increase the protective effect of murine nerve growth factor on the retina and optic nerve. Our results show that under the action of high intraocular pressure, the axon structure of rabbit optic nerve is disordered, the myelin sheath is dissolved and loosened to varying degrees, and the microtubule and microfilament structure disappear. After injection of the mouse nerve growth factor through the vitreous cavity, the number of optic nerve axons increased, the diameter of the optic nerve axon decreased, and the percentage of the optic nerve axon accounted for the cross-sectional area of the optic nerve increased. In the ultrasound microbubbles combined with the mouse nerve growth factor group, the axonal swelling was significantly reduced, the diameter of the axons was reduced, the degree of loss of myelin in the optic nerve axon was significantly reduced, and the number of axons was increased, which was statistically significant compared with the other groups. P < 0.05). In addition, this study also observed the pathological histomorphology and ultrastructure of the retina and optic nerve. It was found that the ultrasound microbubbles combined with the intravitreal injection of the mouse nerve growth factor group had a complete retinal pathological structure, and the layers were clear. The number of RGCs is increased, and even vacuolar-like cells are visible. The ultrastructure of rabbit retina and optic nerve found that the photoreceptor cells of the ultrasound microbubbles combined with intravitreal injection of the mouse nerve growth factor group were slightly disordered, the nuclear membrane of the inner nuclear layer was unclear, and the nucleus membrane of most ganglion cells was clear and the chromatin distribution was uniform. The nucleolus was obvious, and the mitochondria showed no obvious swelling and vacuolization. The optic nerve myelin is densely packed and neat, showing microtubules, microfilaments and other structures. This indicates that ultrasound microbubbles combined with mouse nerve growth factor not only protects RGCs caused by high intraocular pressure, but also protects the photoreceptor layer and the inner and outer nuclear layers.

In summary, ultrasound microbubbles combined with mouse nerve growth factor have obvious optic nerve damage in rabbits with high intraocular pressure. Protective effects. This targeted drug delivery method allows the drug to reach the eye more easily, without systemic metabolism, to avoid the decomposition of the drug through the blood circulation and the interference and destruction process of the drug in the body environment, thereby maximizing It guarantees the concentration of the drug reaching the target organ and exerts the greatest therapeutic effect, which greatly broadens the field of vision of glaucoma optic nerve protection therapy and provides a new idea.

Claims (7)

1. A novel intravitreal injection system for rat nerve growth factor, characterized in that it comprises a mouse nerve growth factor and an ultrasound contrast agent.
2. The drug delivery system of claim 1 wherein said ultrasound contrast agent is sulfur hexafluoride microbubbles.
3. The use of the drug delivery system of claim 1 or 2 for the manufacture of a medicament for the treatment of glaucomatous optic nerve damage for administration via intravitreal injection under ultrasound irradiation.
4. The use according to Claim 3, characterized in that the medicament is administered by intravitreal injection at an ultrasonic sound intensity of 0.5 W/cm ² and an irradiation time of 30 s to 60 s.
5. The use according to Claim 3, characterized in that the drug is administered at a dose of 12 ug/kg of murine nerve growth factor.
6. The use according to claim 3, characterized in that the medicament is a stable suspension of murine nerve growth factor and an ultrasound contrast agent.
7. The use according to claim 6, characterized in that the mouse nerve growth factor is mixed 1:1 with the ultrasound contrast agent in the suspension.

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