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Show ORIGINAL CONTRIBUTION Methylprednisolone Treatment Does Not Influence Axonal Regeneration or Degeneration Following Optic Nerve Injury in the Adult Rat Marcus Ohlsson, MD, PhD, UlfWesterlund, MD, Iver A. Langmoen, MD, PhD, and Mikael Svensson, MD, PhD Background: Methylprednisolone ( MP) is often used to treat optic nerve injury. However, its effects in experimental crush injury have not been extensively evaluated. Methods: Adult Sprague- Dawley rats were subjected to a standardized optic nerve crush injury. Animals were treated either with 30 mg/ kg MP intravenous bolus followed by subcutaneous injections every 6 hours for 48 hours, or with a drug vehicle alone. Results: The injury resulted in a partial loss of neuronal nuclei- labeled retinal neurons and a corresponding degeneration of axons distal to the injury. EDI- labeled macrophages accumulated at the site of lesion, phagocyting FJ-labeled axonal debris. Regenerative fibers expressing growth associated protein- 43 were seen proximal to the lesion, but did not traverse the glial scar. Analysis of optic nerve function using visual evoked potentials showed typical signals in intact animals, which were abolished after injury in MP- treated and untreated animals. Conclusions: We did not detect any effects of MP on retinal cell survival, macrophage activity at the site of injury, axonal degeneration/ regeneration, or visual function. These experimental results provide a physiologic underpinning for the lack of efficacy demonstrated in a large trial of MP treatment of clinical optic nerve injury. ( JNeuro- Ophthalmol 2004; 24: 11- 18) The microstructure of the optic nerve is arranged like other central nervous system ( CNS) neural tracts with astroglial and microglial cells interposed along axons of retinal ganglion cells myelinated by oligodendroglia ( 1). From the Department of Clinical Neuroscience, Section of Neurosurgery, Karolinska Institute and Hospital, Stockholm, Sweden. Address correspondence to Marcus Ohlsson, MD, PhD, Department of Neurosurgery, Karolinska Hospital, 171 76 Stockholm, Sweden. E- mail: marcus. ohlsson@ ks. se. Supported by grants from the Karolinska Institute and the Swedish Medical Research Council. Axotomy of these nerve fibers leads to anterograde Walle-rian degeneration in combination with the formation of a glial scar at the site of injury that blocks regeneration. In contrast to peripheral nerves, an injury to the optic nerve will therefore result in a permanent neurologic deficit with no capacity for spontaneous recovery ( 1- 4). CNS neurons, including retinal ganglion cells, do seem to have regenerative capacity after injury. However, the microenvironment prevents sprouting and axonal elongation beyond the glial scar ( 5,8). Several mechanisms are involved in making CNS tissue impenetrable to growing adult neurites after injury. The glial scar of numerous proliferating astroglial and microglial cells intermingled with axonal and myelin debris creates a growth barrier. In addition, myelin breakdown products contain components that inhibit axonal regrowth ( 6,7). The lack of Schwann cell analogs producing a gradient of appropriate trophic factors in a similar fashion as in peripheral nerves is yet another mechanism likely to be crucial in this complex pathology ( 1). Furthermore, mechanical trauma to CNS tissue leads to migration and accumulation of immune competent cells at the injury site ( 8) and induction of the complement system ( 9- 11). Based on clinical experience with spine trauma, some clinical centers have used methylprednisolone ( MP) as a treatment of optic nerve injury. In 1999, the International Optic Nerve Trauma Study reported observations of 133 patients with traumatic optic nerve neuropathy ( 12). Patients were categorized into three major groups, untreated ( n = 9), corticosteroid- treated ( MP; n = 85), and surgically-treated ( optic canal decompression; n = 33). With the main outcome the measure of visual acuity, the International Optic Nerve Trauma Study reported no clear benefit for either treatment. Using a well- characterized and easily reproducible model of optic nerve injury in adult rats ( 11,13), we have examined whether MP has effects on degeneration, regeneration, macrophage response, or visual function after experimental optic nerve trauma. J Neuro- Ophthalmol, Vol. 24, No. 1, 2004 11 JNeuro- Ophthalmol, Vol. 24, No. 1, 2004 Ohlsson et al. MATERIALS AND METHODS All animal experiments were approved by the local ethics committee at the Karolinska Institute. Adult female Sprague- Dawley rats ( n = 46) weighing 200 g were anesthetized with an intramuscular injection of 0.2 ml Hypnorm ( 0.315 mg/ ml fentanyl citrate and 10 mg/ ml fluanisone; Janssen- Cilag, Ltd., Saunderton, UK) and 0.2- 0.4 mg Dor-micum ( midazolam; AstraZeneca, Sodertalje, Sweden). A supraorbital incision was made, after which the optic nerve was exposed by blunt dissection and microsurgical technique, thus sparing orbital tissue and extraocular eye muscles. The optic nerve was crushed 1 mm behind the globe with a calibrated pair of fine forceps, providing a fixed maximum pressure of 0.6 N for 10 seconds ( 11,13- 14). Care was taken to spare retinal circulation, and the retina was inspected following surgery. Animals with impaired retinal circulation were immediately discarded from further studies. All animals had free access to food and water and were housed according to standard animal care guidelines. Following optic nerve crush, animals were randomized to treatment with MP ( Solu- Medrol, Pharmacia, Uppsala, Sweden; n = 23) or vehicle alone ( n = 23). Animals receiving MP were given a first bolus dose of 30 mg/ kg intravenously in the tail vein 1 hour postoperatively, followed by subcutaneous injections every 6 hours, corresponding to 5.4 mg/ kg/ h for 48 hours and based on current guidelines for clinical treatment of spinal cord injury ( 15). All animals tolerated this treatment well, and no differences in behavior or well- being between MP- treated and untreated animals were noted before sacrifice. Visual evoked potentials were recorded twice weekly preoperatively and postoperatively in twelve additional animals that had received implanted electrodes prior to crush injury. An isolated silver plate electrode was placed extra-durally through a 2 mm diameter craniotomy over the visual cortex using the stereotactic coordinates ( Bregma - 8 mm, lateral 3 mm) based on the Paxinos Atlas ( 16). A reference electrode was placed extracranially, superficial to the olfactory bulbs. A small screw was mounted in the right frontal bone to assure a fixed position. The electrodes were cemented to the skull with dental acrylic resin ( Swebond; Svedia, Enkoping, Sweden). A connector was sutured at the scruff of the animal and further connected to the electrodes via insulated silver wiring. Animals were then randomized to no treatment ( n = 4), MP treatment ( n = 4), or sham treatment ( n = 4). At each VEP reading, the animal was lightly sedated with 1% halothane ( Fluothane; AstraZeneca) and connected to a Grass PolyView recorder ( Astro- Med, West Warwick, RI). Flash stimuli were generated by a Grass PS- 33 Plus Photic Stimulator ( Astro- Med) and evoked potentials were synchronized, recorded, and further processed using Grass PolyView 2.5 ( Astro- Med) software. Baseline recordings of visual evoked potential were recorded 4 days prior to trauma and then repeated twice weekly throughout the study. Because of the electrode arrangement, animals were housed individually and on a 12- hour diurnal cycle. Except for those with implanted electrodes, animals were re- anesthetized at 2, 7, 14, or 28 days after crush and killed by transcardiac perfusion of saline ( 37° C), followed by cold ( 4° C) 4% paraformaldehyde in phosphate- buffered saline ( PBS). The eyes and optic nerves were excised and post-fixed for another 90 minutes using 4% paraformaldehyde in PBS, followed by rinsing in PBS alone. The retinas were rapidly dissected, flat- mounted on microscope slides, air- dried, and processed for immunohistochemistry. Optic nerves were immersed in 15% weight- to- volume ( w/ v) sucrose in PBS overnight, longitudinally cryosectioned ( 14 urn), and mounted on SuperFrost Plus microscope slides ( Menzel- Glaser, Braunschweig, Germany) prior to immunostaining. Animals destined for plastic embedding and semithin sectioning were perfused with 1% glutaraldehyde, 1.15% paraformaldehyde ( w/ v) in a phosphate buffer and post- fixed overnight in the same fixative. Tissue blocks ( 1 mm) of the optic nerves, 6 mm from the site of injury ( proximal to the optic chiasm), were immersed in 1% osmium tetraoxide, dehydrated in a graded series of ethanol to acetone, and embedded in Agar Resin 100. Semithin transverse serial sections ( 0.5 urn) were cut using an Ultratome and stained with toluidine blue. Sections were incubated overnight at + 4° C with primary antibodies ( Table 1). Antibodies were diluted in PBS containing 1% bovine serum albumin and 0.3% Triton X- 100 ( Riedel- deHaen, Seelze, Germany). Routine TABLE 1. Antibodies and marker used in the present study Antibodies and marker Species and type Dilution Source EDI GAP- 43 Fluoro- Jade NeuN mouse monoclonal mouse monoclonal not applicable mouse monoclonal 1: 4000 1: 1000 see Materials and Methods section 1: 500 SerotecMCA341 Boenringer Mannheim Histochem Chemicon MAB377 12 © 2004 Lippincott Williams & Wilkins Methylprednisolone Treatment of Optic Nerve Injury JNeuro- Ophthalmol, Vol. 24, No. 1, 2004 protocols for indirect immunofluorescence ( IF) and avidin-biotin complex ( ABC) techniques were used to visualize the immunoreactivity ( IR). For the ABC technique, sections were rinsed and incubated with biotinylated secondary antibodies ( 1: 200) for 1 hour at room temperature. Sections were then washed in a phosphate buffer and incubated with ABC for 1 hour, after which IR was revealed by incubating sections with diaminobenzidine ( 50 mg/ 100 ml) in Tris- hydrogen chloride buffer ( 0.1 M, pH 7.4) containing 1% ( w/ v) nickel sulphate and 1% ( w/ v) cobalt chloride for 10 minutes, and for another 5 minutes after adding hydrogen peroxide ( 0.02%). Sections were rinsed in Tris and dehydrated through a series of graded ethanols to xylene and mounted on a non- aqueous DPX medium. Secondary antibodies of appropriate species conjugated with Cy3 ( 1: 1000) and diluted in PBS were used for IF- IR. The ABC Elite and the biotinylated secondary antibodies were obtained from Vector Laboratories ( Burlingame, CA), and the diaminobenzidine was obtained from Sigma Chemical Company ( St Louis, MO). For IF, Cy3- or fluorescein isothiocyanate-conjugated complimentary secondary antibodies of appropriate species were obtained from Jackson Laboratory, Inc. ( Baltimore, MD). Fluoro- Jade ( FJ), a marker of degenerating neuronal tissue ( 17) ( Histochem Inc., Jefferson, AR), was diluted to 0.00002% in 0.1% acetic acid. Slides were incubated with FJ solution for 30 minutes. Sections were then rinsed in distilled water, dehydrated, air dried, dipped in xylene, and mounted with DPX as described above. All sections were analyzed using a Leica DMRB epifluores-cence microscope ( Leica Microsystems AG Wetzlar, Ben-sheim, Germany). Arbitrary areas of immunostaining ( EDl- IR) or FJ labeled structures at the site of injury and along the optic nerve, each expressing a homogenous and representative level of labeling, were photographed ( magnification, x40) with a Nikon N90 camera attached to a Leica DMRB microscope ( 10,11,13,18). The uninjured contralateral optic nerve was photographed in all animals and used for comparison between injured and uninjured optic nerves at each separate survival time ( Student t test). Photomicrographs were imported into NIH Image 1.60 software ( NIH, Be-thesda, MD). Following initial calibration and setting of background levels, labeled structures were analyzed with regard to the stained area in pixels using the built- in " density slice tool" in the NIH Image software ( 10,11,13,18). These values, representing pixels, were analyzed statistically using Statistica ( StatSoft, Inc., Tulsa, OK) and Microsoft Excel software ( Microsoft Corp., Redmond, WA). Asterisks indicate level of significance ( Student t test): P < 0.05* ; P< 0.01**. Axons were analyzed by oil- immersion microscopy and magnification x 100 in toluidine blue- stained semithin cross- sections ( 0.5 urn) of optic nerves ( Fig. 2). Axons having a well- defined myelin sheath were regarded as viable, and the number of axons was counted in an area of 10 x 10 \ aa in four non- overlapping areas from each nerve. The average number of axons from these four areas was used as one observation. Retinal neuronal cell bodies labeled with anti-neuronal nuclei ( NeuN) were quantified in the ganglion cell layer and inner nuclear layer in retinal flat- mounts. Labeled structures were counted in one randomly chosen area ( 50 x 50 urn) in each quadrant of the retina, equally distant from the optic nerve head. The mean of NeuN- positive cells from these four areas was defined as one observation. The number of axons, or NeuN- positive cells, was compared between groups at all survival times and statistically analyzed using the Student t test. RESULTS Macrophages- EDl No EDl- IR, a marker for macrophages, was found in sham- operated animals or in intact contralateral optic nerves ( Fig. 1A and B). Very few EDl- positive cells were found at 2 dpi in treated or untreated animals ( not shown). However, the number of EDl- positive macrophages was significantly higher at 7 dpi, and continued to increase at 14 and 28 dpi ( Fig. 1C through G, Fig. 5A). The site of injury was almost totally covered with EDl- positive cells at 28 dpi, clearly contributing to the glial scar formation. Quantification of EDl- IR distal to the site of lesion did not show significant differences between MP- treated and untreated animals ( Fig. 5A). Fluoro- Jade No FJ staining, a marker for degenerating axons, was found in intact optic nerves or sham- operated animals ( Fig. 1A and B). Very few FJ- positive structures were found at 2 dpi in the ipsilateral optic nerve ( not shown). However, at 7, 14, and 28 dpi, numerous FJ- labeled structures interpreted as disintegrating axons appeared at the lesion site and in the distal portion of the nerve ( Fig. 1C through G). Quantification by image analysis showed no significant difference between MP- treated and untreated animals ( Fig. 5B). Quantification of Axons on Semithin Sections The axons in the intact and sham- operated optic nerves appeared to be either relatively large with thick myelin sheaths or small with thin myelin sheaths ( Fig. 2A and B). Numerous degenerative structures, including swollen axons and irregular myelin sheaths, were found distal to the lesion at all survival times ( Fig. 2C through F). However, several axons had intact myelin sheaths on cross- sections, suggesting subtotal injury. No major morphologic differences could be detected between MP- treated animals ( Fig. 2B, D, and F) and untreated animals ( Fig. 2A, C, 13 JNeuro- Ophthalmol, Vol. 24, No. 1, 2004 Ohlsson et al. FIGURE 1. Double- staining of macrophages ( EDI - IR, red) and degenerative axons ( Fluoro- Jade, green). A, B: Intact and sham- operated optic nerves showed no or low FJ staining and EDI - IR. C- H: From 7 to 28 dpi, an accumulation of macrophages is seen at the site and distal to the injury. FJ labeling of degenerative axons shows axonal debris 7- 28 dpi. No obvious differences can be seen between untreated animals ( A, C, E, and G) and MP- treated animals ( B, D, F, and H). Scale Bar: 50 um; Inserts 10 um. and E) without significant differences between groups ( Figs. 2 and 6). A substantial amount of debris was still present in the nerve distal to the lesion at 28 dpi ( Fig 2E and F) in both groups, indicating a relatively slow removal of debris, consistent with the FJ findings ( Fig 1E and F) and findings after injury elsewhere in the CNS. Growth Associated Protein- 43 No growth associated protein ( GAP)- 43, a protein related to a regenerative mode of injured neurons ( 19,20), was found in sham- operated animals or, as was expected, in intact contralateral optic nerves. However, GAP- 43, interpreted as regenerative sprouts, was expressed in neurites proximal to the site of injury at 7, 14, and 28 dpi ( Fig. 3A), but not at 2 dpi. Some GAP- 43- IR structures were also found in the glial scar at the site of injury, confirming results 14 FIGURE 2. Semithin toluidine blue- stained cross sections ( 0.5 um) at the level of the optic canal distal to the site of injury. A, B: Numerous myelinated axons with various diameters were found in sham- operated animals showing no difference between the two groups. C- F: Following optic nerve crush, numerous degenerative profiles were seen distal to the lesion at 7 and 28 dpi. Several intact axons were seen in both groups, suggesting a subtotal injury. Note similar morphology in untreated ( C, E) and MP- treated ( D, F) optic nerves. Scale bar: 10 um. Untreated 28 dpi 50^ MP- treated 28 dpi 50^ FIGURE 3. GAP- 43- IR in optic nerves. A: Low magnification of optic nerve showing GAP43- labeled sprouts proximal to the site of injury ( arrows). B, C: No GAP- 43- IR is detected distal to the site of injury in untreated ( B) or MP-treated animals ( C). Scale bar A: 100 um; B, C: 50 um. © 2004 Lippincott Williams & Wilkins Methylprednisolone Treatment of Optic Nerve Injury JNeuro- Ophthalmol, Vol. 24, No. 1, 2004 C Op, Untreated 21 dpi •|- FIGURE 4. Visual evoked potential readings from implanted cortical electrodes. A, B: Normal preoperative VEP diagrams from intact animal 3 days after electrode implantation ( A), and from a sham- operated animal 21 days post electrode implantation ( B). C, D: The cortical response disappears following optic nerve injury, as expected. Neither untreated animals ( C) nor MP- treated ( D) animals regained a cortical response. obtained in a previous study ( 13). However, no GAP- 43- IR was found distal to the site of injury in MP- treated or untreated animals studied at 2,7,14, or 28 dpi ( Fig. 3B and C). Retinal Neurons Viable neuronal cell bodies in the ganglion cell layer and inner nuclear layer were immunostained with the NeuN antibody ( 21) on retinal flat- mounts. The injury caused a gradual decline of NeuN- labeled retinal neurons over time, corresponding to approximately 10%, 48%, and 75% cell loss at 7, 14, and 28 dpi, respectively ( percentage values refer to the quotient of labeled ipsilateral to contralateral structures) ( Fig. 6). The administration of MP after injury did not influence the number of spared neurons ( Fig. 6). Visual Evoked Potentials Visual evoked potentials from implanted electrodes were successfully read during 3 weeks after optic nerve crush ( Fig. 4). Each animal was compared with its initial baseline recordings and to the data obtained from intact animals. Following axotomy, the cortical response disappeared as expected. Sham- operated animals presented an almost identical cortical response at 21 dpi, compared with the preoperative visual evoked potential ( Fig. 4A and B). Neither untreated nor MP- treated animals regained a cortical response throughout the study. The data showed no differences between MP- treated animals and controls ( Fig. 4C and D). DISCUSSION The optic nerve, like other CNS white matter tracts, fails to regenerate following axotomy. However, previous studies have shown that adult retinal ganglion neurons actually have growth capacity after axotomy if the microenvironment is changed ( 22- 25). This inborn growth capacity has been demonstrated in experimental models using peripheral nerves grafted to the transected optic nerve ( 22,23). Optic nerve regeneration has also been induced by transplantation of cultured macrophages or Schwann cells, thus creating a peripheral nervous system micromilieu ( 8,24,26). Accumulation of macrophages at the site of injury seems to be an important component of the immune response, which may support neuronal repair after CNS injury ( 11,13,26- 28). Although still controversial, patients with acute spinal cord injuries are treated with MP. The mechanism by which MP would protect intact neurons/ axons in a damaged area is not established, but MP is believed to stabilize cell membranes and have the following neuroprotective effects ( 29,30): 1) inhibits development of local edema, thereby minimizing additional damage to neurons/ axons induced by local hydrostatic pressure; 2) inhibits free radical- induced lipid peroxidation; 3) inhibits synthesis of cytokines; 4) maintains tissue blood flow; 5) reduces neurofilament degradation; and 6) improves reversal of intracellular Ca2+ accumulation. Despite the structural differences between the optic nerve and the spinal cord, the two systems correlate well in lesion models ( 31). Treatment with MP has therefore been suggested not only for treatment of spinal cord injury, but also for optic nerve injury. However, due to insufficient evidence, there is no support for specific therapy guidelines. In this study, we have evaluated the effects of MP treatment on optic nerve injury in adult rats using the protocol in clinical use for spinal cord injury. The dose we administered has previously been shown to be about 1000 times the amount necessary to activate glucocorticoid receptors in humans and reduce lipid peroxidation, protect molecular assemblies such as neurofilament synthesis, and reverse lactic acid increase after spinal cord injury ( 32). The crush injury we used creates a subtotal lesion, leaving a fraction of axons uninjured distal to the injury ( Figs. 2 and 6). This experimental model may not reflect the wide variety of clinical optic nerve lesions, but the degree of injury is easy to standardize, making the data reproducible. In line with previous studies using similar experimental methods ( 33), we have confirmed the poor regenerative potential of the optic nerve, even with MP treatment. We found numerous macrophages ( ED1- IR) accumulating at the site of injury, as well as in the distal portion of the optic nerve ( Fig. 1, red), but very few were found proximal to the injury. Quantitative data showed a continuous increase of ED1- IR during the studied period. Despite the intense macrophage activity, a substantial amount of FJ-labeled debris was still present at 28 dpi ( Fig. 1, green) in 15 JNeuro- Ophthalmol, Vol. 24, No. 1, 2004 Ohlsson et al. Macrophages ( ED1- IR) 70000 ,- s 60000 tn 3 X soooo ( Q 40000 o ™ 30000 ? Bars from left to right: • Op, Untreated DOp, MP- treated fflUnop, Untreated lUnop, MP- treated Sham 2 dpi 7 dpi 14 dpi Fluoro- Jade 50000 45000 ^-^ 40000 tn J£ 35000 • S 30000 £ J 25000 "( O0 20000 • Op, Untreated DOp, MP- treated UUnop, Untreated HUnop, MP- treated 28 dpi FIGURE 5. A) Quantification of ED1- IR macrophages and B) quantification of F- J-stained degenerating optic nerve axons. There is an increase in EDI - IR and Fluoro- Jade labeling in operated optic nerves in comparison to unoperated nerves. However, there were no significant differences between untreated and MP- treated animals. ( Student f test, P > 0.05). OP = operated ( optic nerve crush injury). Sham 2 dpi 7 dpi 14 dpi 28 dpi the entire distal portion of the nerve, which was confirmed by the morphology in semithin sections ( Fig. 2). In addition, large EDI- positive macrophages with FJ- labeled phagosomes were found at 28 dpi ( Fig. IF and G inserts). Our data show an intense response of phagocytic macrophages, but also that these cells lack capacity to remove axonal and myelin debris as fast as in the peripheral nervous system, which may explain the poor growth of axons in the injured CNS. Viable retinal neural cells and optic nerve axons declined with longer survival times, regardless of treatment strategy ( Fig. 6). In our experiment, MP treatment failed to change any of the studied parameters of degenerative or regenerative events in the optic nerve. VEP analysis showed no signs of functional recovery following MP treatment. We cannot, of course, exclude small differences in optic nerve function between groups that are not reliably differentiated with VEP. The results obtained in this experimental study provide a physiologic underpinning for the lack of efficacy of MP in the treatment of clinical optic nerve injury found in the International Optic Nerve Trauma Study ( 12). 16 © 2004 Lippincott Williams & Wilkins Methylprednisolone Treatment of Optic Nerve Injury JNeuro- Ophthalmol, Vol. 24, No. 1, 2004 Quantification of NeuN- IR ( neurons) in the retina B Sham 2 dpi 7 dpi 14 dpi 28 dpi Quantification of axonal numbers Bars from left to right: • Op, Untreated DOp, MP- treated • Unop, Untreated • Unop, MP- treated Bars from left to right: • Op, Untreated • Op, MP- treated • Unop, Untreated • Unop, MP- treated FIGURE 6. Quantification of retinal neurons ( NeuN- IR; A) and optic nerve axons ( B). With longer survival times, the number of viable neurons in the retina and the number of intact optic nerve axons decreases, but without significant differences between untreated or MP- treated animals ( Student f test, P > 0.05). 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