WO1990005481A1

WO1990005481A1 - In vivo window chamber

Info

Publication number: WO1990005481A1
Application number: PCT/US1989/005110
Authority: WO
Inventors: Walter J. Levy, Jr.
Original assignee: Levy Walter J Jr
Priority date: 1988-11-14
Filing date: 1989-11-14
Publication date: 1990-05-31
Also published as: CA2002891A1

Abstract

Apparatus for in vivo optical visualization of a biological process of a living tissue includes a chamber (10) having a bottom supporting member (14) and a top transparent member (12) spaced apart from the bottom member (14) and sealed to the bottom member (14) at a portion of its periphery with a biologically inert material, and having a port (16, 18, 20) into which the living tissue extends.

Description

■ IN VIVO WINDOW CHAMBER

Background of the Invention This invention relates to visualizing (i.e. , observing, by eye or using a microscope) cells and cell structures in vivo.

Observation of biological and nervous system tissue structure and function has been done on whole organs and histological examination has been done of terminal tissue from sacrificed animals. Individual cells capable of growth and cell division also have been investigated for limited periods of time in vitro by culturing the cells in media designed to mimic the natural physiologic state.

Clark et al . (1933), Anatomical Record, Vol. 12, pp. 105 et seq. , describes implanting a chamber in the ear of a rabbi-, and permitting connective tissue to regrow around the chamber to study effects of drugs on blood flow. The chamber included two layers of mica or glass into which the tissue regenerated, allowing visualization of the chamber contents when implanted in a hole in the rabbit ear. undberg et al . (1982), J. Neuropath. Expt. , Vol. 4, pp. 412 et seq. , sectioned a nerve of an animal, implanted a εilicone tube nerve chamber in the nerve, and then allowed the nerve to regrow to study nerve tissue regeneration. The chamber was a cylindrical nontransparent tube, approximately the diameter of the nerve, that was placed around and between the nerve stumps. Lichtman et al. (1987), J. Neuroscience,

Vol. 7, pp. 1215 et seq. , attempted to directly visualize selected cells by direct visualization of surgically exposed neural structures, e.g. , the cervical sympathetic chain, using flourescent dyes injected into an animal. Other methods of tissue visualization include implanting a piece of glass over a portion of exposed brain tissue and observing tissue function without allowing tissue regrowth.

Summary of the Invention The invention allows, for the first time, high resolution in vivo observation of cellular events involved in tissue function, including nerve regeneration and blood flow. The invention features, in one aspect, an apparatus for in vivo visualization of a biological process of a living tissue. The apparatus includes a chamber having a bottom supporting member and a top transparent member that is spaced apart from the bottom member and sealed to the bottom member with a biologically inert material. The chamber has a port into which the living tissue extends.

In preferred embodiments, the apparatus can be used to visualize simultaneously the biological processes of neural tissue regeneration and blood flow. Preferably, the top and bottom members are spaced in parallel orientation; the space between the walls of the chamber is at minimum wide enough to allow tissue regrowth, and narrow enough to allow microscopic imaging; the distance between the walls is preferably between 40 and 250 microns, and most preferably 100 microns. A microscope can be positioned onto or near to the top member of the chamber for visualizing the tissue. Preferably, the top member of the chamber is removable, to open the chamber and provide access for instrumentation, introduction of chemical substances, or transplatation of tissues. Most preferably, both the walls of the chamber and the removable top are made from high optical quality glass, such as microscope glass coverεlips. Preferably, the parallel pieces of the chamber are sealed at their periphery with a biologically inert material; and the chamber includes one of more ports, i.e., openings, which are approximately 1 mm in diameter and through which tissue can grow; each port may have attached to it a sleeve of inert material; preferably the biologically inert material sealing the edges and forming the port sleeves is made of silicone elastomer.

The chamber of the invention is used in a method for visualizing a biological process of living tissue in vivo, by inserting the chamber into living mammalian tissue, positioning a microscope to view into the chamber, and visualizing the tissue through the microscope.

The apparatus is particularly useful for visualization of neural tissue, such as transplanted fetal brain tissue. The biological process visualized by the invention can be a single process, or the method can be used for simultaneous visualization of any or all of the following: tissue regeneration, blood flow, cellular growth, a measurable biochemical function, or axonal transport. Preferably, the microscope inserted onto the chamber is an operating microscope, or a high resolution microscope that allows either epiillumination or tranεillumination with nomarski optics and video image enhancement. Preferably, the chamber is filled with saline, or with a growth factor such as nerve growth factor or platelet derived growth factor, or with a trophic factor such as brain derived neurotrophic factor, or with a drug; these substances can be studied for their effect on biological functions of the tissue. The invention allows for the first time direct simultaneous dynamic study of living neural and vascular tissues, e.g. , nerve tissue, in vivo. Blood flow can be visualized in vessels growing inside the chamber under selected conditions, and qualitative changes in flow through vessels can be observed at a selected resolution. Thus, changes that occur in cell structure or function as a result of blood flow changes can also be observed and recorded.

Other features and advantages of the invention will be apparent from the following description of the preferred embodiments thereof, and from the claims.

Preferred Embodiments We turn now to a description of preferred embodiments of the invention, after first briefly describing the drawings. Drawings

Fig. 1 is a diagrammatic representation (plan and side views, 1.1, 1.2) of a chamber of the invention in a rectangular (e.g. , square) configuration.

Fig. 2 is an illustration (plan and side views, 2.1, 2.2) of a chamber in a round (e.g. , circular) configuration with a removable top, in place in the sciatic nerve of an animal.

Fig. 3 is a diagrammatic representation (side view) of a chamber in place in a spinal cord of an animal, where it is installed around the remaining cord, which has been surgically thinned. Fig. 4 is a photograph of the cellular contents of a chamber in place in a severed nerve, showing the nerve stumps regrowing into it.

Fig. 5 (5.1-5.6) is a sequential series of photographs of a chamber in place in a nerve, illustrating axonal transport in a single axon using high resolution video enhancement techniques on the chamber.

The following is a description of the invention and its use. Construction of Chamber

In one embodiment, shown in Fig. 1, the apparatus includes a rectangular (square) implantable chamber 10 which consists of two 5mm square surfaces, top 12 and bottom 14, either or both made from optically high quality transparent material, such as microscope coverslip glass (obtained from Thomas Scientific, Swedeεboro, N.J.). Glass walls are preferable because tissue does not adhere to glass; alternatively, plastic, or another transparent material may be used. The top and bottom wall surfaces 13, 15 delimiting the space 11 within the chamber are separated by a spacer made of glass or plastic. The surfaces are approximately 200 μm apart, but may be between 50 and 250 microns apart, a distance which is thin enough to allow observation with a microscope system, and thick enough to allow cellular survival and growth.

The surfaces of the chamber are sealed at the edges by a substance that is biologically non-reactive, such as silicone elaεromer (Dow Corning, MDX , 4210). Gaps in the sealed edges serve as ports 16, 18, and 20 for the inclusion of neural εyεtem or other tissue in passage, or for the ingrowth of regenerating tissue through the chamber . The ports are between 0.5 and l.0 mm in diameter and lead into the chamber through a biologically nonreactive entryway or cuff, also of silicone elastomer; silastic tubeε (ID = 0.76 mm, OD = 1.6 mm) may be used as cuffs to hold the nerve stumps in place. Two tubes 17, 19 may be placed at the midpoints of opposing sides of the chamber; in addition, a third tube 21 can be placed at the midpoint of a third side and plugged with a tightly fitting silastic tube. The tisεue may be secured to the cuff by means such as a suture when the tisεue is inserted into the chamber . The cuffs also help prevent non-nerve tissue material from entering the chamber. In one embodiment, the chamber volume is 2.5-3.0 mm .

Fig. 2 shows a chamber 110 having a round (circular) configuration in plan view and having a removable top (lid) 122 made of high resolution, optical ■quality material (glass). The lid is sealed to the chamber body 114 by means such as a silicone elastomer gasket to prevent ingrowth of material, e.g., non-nerve material, into the space 111 within the chamber. The lid can be repeatedly removed and replaced, by breaking the seal, without harm to the chamber contents. Removal of the lid also allows material to be inserted into the chamber, such as chemical substances or biological tisεues (e.g., cells from tissue culture or transplanted cells). Removal of the lid also allows material to be inserted into the chamber, such as chemical substances or biological tissues (e.g., cells from tissue culture or transplanted cells) . Removal of the lid alεo allows selected recording and instrumentation of the chamber by electrophyεiological or biochemical recording devices. The silicone reseals when the lid is replaced. Placing the Chamber In Vivo

In order to place the chamber into the desired tissue of an animal, the animal is first anesthetized using, e.g. , phenobarbital at an optimal dosage, or any conventional anesthetic. A linear incision is then made in the area of interest, and, where the tissue to be observed is a nerve, the nerve is diεεected free of surrounding tissue until it is exposed. The nerve is then severed and the stumps are inserted each into a ports of the chamber. By way of example. Fig. 3 shows the chamber, including lid 212 and bottom 214, in place in the spinal cord of an animal, and Fig. 4 shows the nerve εtumps growing into the chamber. The wound is then closed and allowed to heal for a time. The time for healing depends generally upon the size of the chamber, and the kind of biological events to be visualized. For mammalian nerve regeneration through a circular chamber of the invention having a diameter about 10 mm for example, the initial ingrowth into the chamber can take about three weeks to heal; for a smaller size chamber, a shorter length of time can be required. During the healing time, the tissue will regenerate and vessels will grow into the chamber through the ports. Although it is posεible to leave the wound open, it is often preferable to close the wound to allow tissue growth, At varying intervals, the animal may again be anesthetized and the wound reopened to accesε the chamber. It can be desirable to acceεε the chamber for any of a number of reasons: to record the electrical activity of nerve tiεsue, to inject subεtanceε into the chamber, to withdraw fluids or other tissues, to manipulate the contents, e.g. , to create lesions in the tiεεue, or to transplant tissue. Substanceε that may be injected into the chamber in order to observe the effect on tissue regeneration include growth factors such as nerve growth factor, platelet derived growth factor, laminen, or type IV collagen. Tissues that my be transplanted into the chamber include fetal brain tiεsue, spinal cord tissue, dorsal root ganglion, peripheral nerve tiεsue, autonomic nervous syεtem tiεεue, vaεcular beds of tissue, or any other transplantable tiεεue. Viεualization

In order to viεualize biological proceεεes, e.g. , cellular functions such as tissue regeneration and blood flow, an epiilluminating operating microscope may be used to see tissue at a magnification of approximately 40X. An epiilluminating syεtem is preferred because it allows objects to be viewed from the same side of the specimen as the viewing optics. Alternately, a transilluminating light source may be used; this allows light to be transmitted through the specimen. An example of an operating microscope that can be used according to the invention is Zeisε Opmi VI from Inεtrument Associateε, Inc. (Longwood, FL) .

Biological processes can be visualized at a higher level of magnification using an AVEC-DIC (Allen Video Enhanced Contrast - Differential Interference Contrast) microscope system (Nikon Optiphot, Southern Micro Instrumentε, Atlanta, Georgia). Thiε magnification level is to approximately 12Q0X and allows visualization of cells and subcellular organelles and processes. In addition, for visualization of cell structure, the video enhanced contrast and differential interference contrast allows magnification to a level normally seen only be electron microscopy. A Nikon Optiphot with rectified Nomarski optics, a Dage MMI model 67 video camera with a Novican tube, a Dage television monitor and a Panasonic video 3/4" recorder may be uεed for cell εtructure imaging. The Nomarεki optics provide substantially improved contrast and edge enhancement, as doeε the computer image proceεεing. Thiε improveε the ability of the human eye to see and recognize objects. Fig. 5 shows an example of axon transport visualized (arrows) in the chamber. Alternatively, a confocal microεcope, uεing a laεer beam through the lenε to improve the image further, could be used. Thiε equipment iε also available from Southern Micro Instrumentε. Placing Chamber in Sciatic Nerve of Rat

The growth of rat peroneal nerve tiεεue waε viεualized uεing a rat sciatic nerve in a chamber having top and bottom surfaces made from coverεlips. Female Sprague-Dawley rats, weighing 250-280 gm, were anesthetized with sodium pentobarbital (50 mg/kg) . The sciatic nerve was exposed in the right mid-thigh and the peroneal branch separated with its blood supply preserved. The nerve was first sectioned under an operating microscope with a surgical knife blade or fine sciεsorε, and the two ends of the peroneal branch were then inserted into the cuffs of the chamber and sutured using 10-0 Ethilon sutures. One suture was tied through the perineurium just behind the stump end and served to hold the stump closely against the gap between the coverslips. A second suture was tied through the perineurium at the end of the port to prevent tranεmisεion of tension to the end of the stump. A silastic tube slit lengthwise waε attached to the bottom of the chamber, and placed around the intact tibial branch of the sciatic nerve. Thiε helped to prevent excessive movement of the chamber and also minimized tension on the stumps. The chamber was pre-filled with either sterile saline or Matrigel (10 mg/ml), a type IV collagen, laminin and heparan sulphate proteoglycan mixture having a composition similar to basal lamina. The wound was flushed with sterile saline and a penicillin streptomycin mixture (Combiotic, Pfizer, Groton, Conn.), and then closed in layers. So placed, the chamber could readily be examined by re-anaesthetizing the animal and opening the wound.

A total of 25 animals were implanted as described above with saline filled chambers and another 15 animals were implanted with Matrigel filled chamberε. In 4 more animalε, the distal port waε εealed with a tightly fitting silastic 'stopper', and the distal nerve stump was resected back to about 5 mm from the port, and the end tied off. The tissue growth into the chambers was observed and photographed periodically with the operating microscope. At 8, 11, 15 and 20 days, two chambers from each series were prepared for electron microscopic (EM) examination. These animals were perfused, under deep pentobarbital anaesthesia, with 0.9% saline and then 2% paraformaldehyde, 2.5% glutaraldehyde in 0.1 M phosphate buffer (PB) , pH 7.4. The chamber was then removed, gently opened and the contents immerεion fixed overnight in the above fixative at 4°C. The tissue was then post-fixed in 1% oεmium, dehydrated through a graded serieε of alcoholε, placed in propylene oxide and embedded in Epon. Sectionε were examined on a Zeiεε EM 10 electron microεcope. In εelected animals, the chamber contentε were fixed in freεhly prepared 4% paraformaldehyde in 0.1 M phoεphate buffer pH 7.4., examined with rectified Nomarεki microεcopy, and then stained either with a monoclonal anti-neu ofilament antibody (e.g., Sternberger-Meyer, Molecular Probeε, Eugene, Oregon) to εhow the axonε or with DII (l, 1'-dioctadecy1-3,3,3' ,3 '-tetramethylindo- carbocyanine perchlorate, Molecular Probeε), a lipid εoluble carbocyanin dye which randomly εtained the entire cell membrane of variouε cells. For antibody staining, the entire proximal fan was washed in several changes of 0.9% saline in 0.1 M phoεphate buffer pH 7.4 (PBS). The tissue was then incubated in 10% normal rabbit serum (KRS) , 0.3% Triton X-100 in PB for two hours at room temperature and then in a 1:50 dilution of a monoclonal antibody againεt rat brain neurofilamentε (e.g., Sternberger-Meyer, SMI 33, Molecular Probeε) with 5% NRS and 0.3% Triton X-100 in PBS for 2 days at 4°C. After washing in three changes of PBS the tissue was incubated in 1:25 FITC labelled rabbit anti-mouse immunoglobulin antibody with 0.3% Triton X-100 for 1 day at 4°C. The tissue was then washed in PBS and mounted in glycerol/PBS, 9:1, and examined with a fluorescence microscope.

Small crystalε of DII were placed directly on the fixed tissue and left in fixative for several weeks. The whole piece of tisεue waε then mounted in glycerol/PBS, 9:1, and examined through the rhodamine filter on a floureεcence microscope.

Great care waε taken to ensure that the initial conditions were identical for each chamber implanted. When this was not possible any variable factors were noted. Two principle variable features were, first, in some chambers the nerve stumps were not as well apposed to the chamber opening as in others and, second, some chambers filled with blood within 30 minutes of implantation while others did not. Neither of these variables resulted in differences in growth rates. Presumably exact alignment of the εtumpε with the chamber waε not critical. In all cases there waε evidence of considerable remodelling of the tip of the εtump, which generally formed a tapered appearance, and this may have tended to equalize the starting situation. The chambers were uεually filled with a clear, εtraw colored fluid at 6-8 dayε whether or not bleeding into the chamber had occurred initially. Examination with the operating microscope revealed that tissue had entered the chamber by 6-8 days in most cases. Growth occurred from both the distal and proximal stumpε simultaneously. Probing with a fine tungsten needle through a side port revealed that a relatively solid fibrin matrix filled the entire chamber at this stage. The ingrowth from both stumps consiεted of a semi-tranεparent material which contained finely branching blood vessels and usually had a band of extravasated blood around the edge. The tissue extended 1.0-1.5 mm into the chamber by 11 days and formed a continuous bridge by 14-16 days.

There was no definite directionality to the tiεεue growth from either stump. The blood vessels and other tissue elements usually extended across the entire width of the chamber before advancing very far acrosε the chamber. The curved growth front alεo εuggeεtε equal extenεion in all directions, although a larger chamber would be required to determine if this pattern would have continued since the chamber walls restricted lateral growth. At later stageε the blood veεεelε started to mature in the region closest to the stumps and a vigorous blood flow could be observed in the loops. Arterioles with narrow lumenε and rapid blood flow could be diεtinguiεhed from venuleε which had larger lumenε and low velocity blood flow. In theεe more mature regions, the tisεue became confined to the interεtump axiε although the growth front waε still widespread, resulting in a fan shaped appearance. When the two 'fans' met at 14-16 dayε many blood vesselε linked up and established a longitudinal blood flow through the chamber. The tissue bridge εubεequently remodelled to be the same width as the nerve εtump throughout the chamber.

At 8 dayε, EM revealed axonε among the fibroblasts, Schwann cells, macrophages and endothelial cells of the proximal growth. The axons were always εurrounded by cellular proceεεeε and never free in the extracellular space, which waε conεiderable at thiε stage and filled with an amorphous material and some fibrin (recognizable by its characteristic structure). Despite this cloεe association with the cells, the highly organized regeneration units described after nerve crush, cut or cylindrical chamber implants were not particularly evident. Many large profiles containing dense aggregations of highly branched tubules, occasional mitochondria and large vesicles were observed with groups of axons particularly after a continuous bridge had formed. The absence of an accumulation of mitochondria or vesicles containing degenerating material suggests that these are axomal growths cones rather than blocked or retracting axonε. Even at 20 dayε (approximately 5 dayε after continuous bridge formation) many immature features were found.

Many growth cones were found in the proximal part of the bridge along with multivesicular artifacts characteristic of newly formed membrane with a high lipid content. In some cases, there was an obvious difference in the maturity of the proximal and distal regions of the bridge. The distal bridge contained a higher proportion of cellular .elements and had more immature blood veεεelε than the proximal εtump. In general, at early stageε and closer to the fan tip, the blood veεselε εhowed many εigns of immaturity. The endothelial cellε contained extenεive rough endoplaεmic reticulum, were very actively pinocytoεing from both surfaces, and were only just forming tight junctions. At 100 days, most vesselε had tight junctionε but εtill had εome immature featureε. Examination of the chamber content with the AVEC-DIC εystem, and in one case with a monoclonal antibody to neurofilaments, showed disorganized and widespread axonal growth, many axonal pathways were tortuous and even U-bends were quite common. Several clear exampleε of axonal branching were found but an estimate of the frequency of branching could not be made.

Many of the cells in the fan-like growth had morphologies reminiscent of specific cell types grown in tissue culture. Random labelling with DII revealed fibroblast-like cells with extensive flat procesεeε and fine surface protrusions. Elongated cells arranged in rows, also with fine processes, are characteristics of Schwann cells as seen in culture.

In the majority of the 100 day chambers, the growth had remodelled to form a compact bridge between the stumpε of even width. The axons were arranged in fascicleε and wrapped by Schwann cellε with a definite basal lamina and, in many caεes, well formed myelin εheaths. Thin, non reactive cellε formed a perineurial εheath and the blood veεεelε had mature tight junctions. Occasionally, an arm connecting the bridge to the chamber side was retained. Osmium tetroxide staining of the myelin εheaths showed that mature axons entered the bridge and then looped around in order to exit and reach the distal stump. Theεe εide arms were alεo wrapped in a perineurium and εupplied with blood vessels . Many preliminary chambers deεignε have been examined and obεervationε εuggeεt that the εide arm retention doeε not correlate with chamber thickneεε or orientation but rather with the location of rough surfaces which presumably allow strong attachment of fibroblasts. The examination of the ultrastructure of chamber contents at 100 days after implantation in the rat peroneal nerve demonstrates axonε with normal morphology, extenεive myelination, mature blood veεεelε and a perineurial sheath. Very few degenerating axons were found and therefore we conclude that confinement within a narrow sealed chamber does not have any major deleterious effects on peripheral nerve regeneration. Therefore, the advantage of high resolution visualization of the contents afforded by this chamber design has been achieved without major diεruption to the eventε being εtudied.

Growth in Absence of Distal Stump: The growth of the proximal nerve stump into the chamber was very similar to that described above even when the distal stump was not present. The growth reached at least to the center of the chamber in each case. At 20 dayε after implant, there were mature blood veεεelε, myelinated axons and compact, aligned Schwann cells and fibroblasts in the proximal part of the fan. There was a progresεion to leεs mature veεεelε, a leεε compact and aligned cellular arrangement and leεs well defined perineurium towards the tip of the growth. Myelinated axons extended 1 mm into the fan. After 20 days, there waε a progressive withdrawal of the ingrowth which resulted in a chamber filled only with clear fluid.

Growth Rates/Effect of Gel: Initial growth rates for bridging of the chamber were calculated by recording the diεtance from the nerve εtump to the tip of the fan-like ingrowth at εucceεεive εtageε in individual animals. EM showed a close relationship between axonal and cellular growth and, therefore, these rates reflect the rate of axonal bridging. In saline pre-filled chambers the proximal growth rate was 0.32 ± 0.06 mm/day (mean with twice the standard error, n = 13) while the distal rate waε 0.18 ± 0.06 mm/day (n - 12). Thuε, the distal growth rate was significantly slower than the proximal rate at 8-11 dayε after implant (p< 0.002, unpaired t-teεt, two tailed). Examination of the absolute ingrowth distance at each time point revealed, however, that the distal stump grew in either earlier or more rapidly and, therefore, the slow growth rate represents a reduction over initial growth. The proximal growth rate was unaffected by absence of the distal stump (the growth rate from 8 to 11 days waε 0N38 ± 0.10 mm/day, n == 4) and therefore the distal stump does not seem to accelerate the rate of growth although it is required for maintenance of the proximal fan. The average proximal growth rate for Matrigel pre-filled chambers (0.46 ± 0.14 mm/day, n = 13) was slightly higher and more variable than the saline chamber rate but the difference was not significant (p< 0.05, unpaired t-test, two tailed). These reεultε εhow that although nerve tiεsue in chambers containing the type IV collagen/laminin matrigel did have a faster regeneration rate than that present in the saline chambers, the rates of tissue regeneration in the chambers did not differ by the significant degree. This suggests that there is lesε of an advantage for thiε biological subεtrate in vivo that in tiεsue culture.

The structure of the growth in the gel chambers waε εimilar to that of the saline chambers at early stageε except that the endoneurium often contained cellular regions associated with extracellular matrix. Within the 11 chambers examined qualitatively at 100 dayε, there was a range of structures. All bridges had a perineurium surrounding a central core where the majority of axons were located. However, the number and organization of the myelinated axons varied conεiderably between chamberε. The major determinant of εtructure appeared to be the thickness of the bridge (the bridges varied in theickneεε from 81 μm to 217 μm with the majority being around lOOμm; all valueε determined in 1-2 μm plastic sectionε) . Thus, both εaline and matrigel pre-filled chamberε with thickneεεeε greater than lOOμm had a diεtinct core composed largely of axons and accompanying Schwann cells, in thinner bridges, here appeared to be retention of perineurial thicknesε at the expense of the axonal core. In these cases, axons were often isolated to islands between the perineurial cells. Qualitatively this appears also to result in a greater proportion of large endoneurial blood vessels at 100 days.

No axons were found growing freely in the Matrigel without cellular contact. In general, however, axonal elongation is partially dependent on Schwann cell contact even in the presence of baεal lamina. Thuε, if Schwann cells do not migrate faεter in the gel, the lack of an enhancement may be explained. Sice the preεence of type IV collagen and laminin iε more conductive to Schwann cell, endothelial, cell and fibroblast differentiation in culture, a similar effect in vivo would inhibit cell division and migration and, therefore, slow regeneration. Placing Chamber Around Spinal Cord

Spinal cord regeneration was studied using the chamber. The chamber waε placed around the εpinal cord aε follows. A Sprague-Dawley rat, approximately 250 gmε, was anesthetized as described above and in incision was made in the upper thoracic area, overlying the spinal cord. Once the spinal cord was exposed in a standard fashion, it was surgically pared to the dimension, e.g., 1-2 mm of neural cord, including the anterior εpinal artery, which could be accommodated in the chamber without injury to the tisεue. In thiε application the chamber iε asεembled around the εpinal cord εo that the tiεεue of the εpinal cord is left in continuity (Fig. 3) . The wound was then sutured closed and allowed to heal for a minimum of one week.

After 2 weeks, the wound was re-opened for inspection of spinal cord regeneration. An epiilluminating microscope (Nikon Optiphot with epiilluminating optics and fluorescence) was positioned so as to visualize the uppermost axons in the chamber and the concomitant blood flow. The animals with the chamber implanted were observed for up to three months following implantation and showed recovery of neurological function, e.g., ambulation. Thus, axonal function can be studied directly over long periods of tissue regeneration under selected conditions.

Other Embodiments Other embodiments are within the following claims. For example, the apparatuε can alεo be utilized for visualizing tissue other than neural tisεue, such as muεcle, omentum, vascular tissue, or that of other organε. The apparatuε can alεo be used for research on the effect of chemicalε on tiεεue regeneration; to test parameterε such aε optimal doεe and order of uεe of drugs, for example, for adminiεtration of methotrexate and interferon for cancer therapy; to determine the timing and dosages of chemicals such as those uεed in fluoreεcence microscopy; to measure physiological parameters within the chamber, such as pH or Ca++ concentration; and to use voltage sensitive or biochemically sensitive dyes.

It may be desirable to inject drugs into an animal, or to add them directly to the chamber, or to use a dialysis tube, or slow-release compound. It is possible to introduce drugs through the ports into the chamber, or through the removable lid. What is claimed is:

Claims

Claims 1. Apparatus for jLn vivo optical visualization of a biological process of a living tissue, said apparatuε compriεing a chamber comprising a bottom supporting member and a top transparent member spaced apart from said bottom member by a distance to provide a chamber space and sealed to said bottom member at a portion of its periphery by a biologically inert material, said chamber having a port into which said living tisεue extends. 2. The appareatus of claim 1 wherein at leaεt a portion of said top transparent member is constructed of high optical material. 3. The apparatus of claim 1 or 2, said bottom and top members being spaced parallel. 4. The apparatus of claim 2, said high optical quality material being glass. 5. The apparatus of claim 2, said high optical quality material being plastic. 6. The apparatus of claim 1, said chamber space being narrow enough to allow microεcopic viεualization and wide enough to allow tissue regrowth. 7. The apparatus of claim 6, said distance being between 40 and 250 microns. 8. The apparatus of claim 7, said distance being 100 microns. 9. The apparatus of claim 1, said biologically nert materiual being silicone elastomer. 10. The apparatus of claim 1, said port being approximately 1 mm in diameter and made of biologically inert material. 11. The apparatus of claim 10, said biologically inert material being silicone elastomer. 12. The apparatus of claim 1 wherein said top is removable to permit opening said chamber without diεrupting the biological procesε and to allow introduction of instrumentation or chemical substances, or transplantation of tissues. 13. The apparatus of claim 12 wherein said removable top comprises a high optical quality glass microscope coverslip. 14. The apparatus of claim 1, for use in visualizing neural tissue. 15. The apparatus of claim 14, said biological procesε visualized being the simultaneous observation of neural tisεue regeneration and blood flow. 16. A method of visualizing a biological proceεε of a living tissue in vivo, said method comprising inserting the apparatus of claim 1 into mammalian tissue, positioning a microscope to view into said chamber, and visualizing the tissue through the microscope. 17. The method of claim 16, for use in visualizing any or all of the biological proceεseε of tisεue regeneration, blood flow, cellular growth, and axonal transport. 18. The method of claim 17, for use in visualizing neural tissue. 19. The method of claim 17, for use in visualizing transplanted tissue. 20. The method of claim 18, said transplanted tissue.being fetal brain tissue. 21. The method of claim 16, for use in conjunction with an operating microscope. 22. The method of claim 16, for use in conjunction with a resolution microscope allowing epiillumination. 23. The method of claim 16, for use in conjunction with a resolution microscope allowing transillumination with nomarski optics and video image enhancement. 24. The method of claim 16, said chamber being filled with a chemical. 25. The method of claim 24, said chemical being saline, a drug, a growth factor, or a trophic factor. 26. The method of claim 25, said growth factor being nerve growth factor or platelet derived growth factor. 27. The method of claim 26, said trophic factor being brain derived neurotrophic factor.