Myelin Sheath Area Research Articles

Cerebral Perfusion Characteristics and Dynamic Brain Structural Changes in Stroke-Prone Renovascular Hypertensive Rats: A Preclinical Model for Cerebral Small Vessel Disease.

Hypertension is a leading cause of cerebral small vessel disease (CSVD) and vascular dementia in elderly individuals. We aimed to assess cerebral perfusion and dynamic changes in brain structure in stroke-prone renovascular hypertensive rats (RHRSPs) with different durations of hypertension and to investigate whether they have pathophysiological features similar to those of humans with CSVD.The RHRSP model was established using the two-kidney, two-clip (2k2c) method, and the Morris water maze (MWM) test, MRI, immunohistochemistry, and biochemical analysis were performed at multiple time points for up to six months following the 2k2c operation.Systolic blood pressure was significantly greater in the RHRSP group than in the sham-operated group at week 4 post-surgery and continued to increase over time, leading to cognitive decline by week 20. Arterial spin labeling revealed cerebral hypoperfusion in the RHRSP group at 8 weeks, accompanied by vascular remodeling and decreased vessel density. Diffusion tensor imaging and Luxol fast blue staining indicated that white matter disintegration and demyelination gradually progressed in the corpus callosum and that myelin basic protein levels decreased. Eight weeks after surgery, blood-brain barrier (BBB) leakage into the corpus callosum was observed. The albumin leakage area was negatively correlated with the myelin sheath area (r=-0.88, p<0.001). RNA-seq analysis revealed downregulation of most angiogenic genes and upregulation of antiangiogenic genes in the corpus callosum of RHRSPs 24 weeks after surgery.RHRSPs developed cerebral hypoperfusion, BBB disruption, spontaneous white matter damage, and cognitive impairment as the duration of hypertension increased. RHRSPs share behavioral and neuropathological characteristics with CSVD patients, making them suitable animal models for preclinical trials related to CSVD.

An injectable, self-healing, electroconductive hydrogel loaded with neural stem cells and donepezil for enhancing local therapy effect of spinal cord injury

BackgroundSpinal cord injury (SCI) is a serious injury with high mortality and disability rates, and there is no effective treatment at present. It has been reported that some treatments, such as drug intervention and stem cell transplantation have positive effects in promoting neurological recovery. Although those treatments are effective for nerve regeneration, many drawbacks, such as low stem cell survival rates and side effects caused by systemic medication, have limited their development. In recent years, injectable hydrogel materials have been widely used in tissue engineering due to their good biocompatibility, biodegradability, controllable properties, and low invasiveness. The treatment strategy of injectable hydrogels combined with stem cells or drugs has made some progress in SCI repair, showing the potential to overcome the drawbacks of traditional drugs and stem cell therapy.MethodsIn this study, a novel injectable electroactive hydrogel (NGP) based on sodium hyaluronate oxide (SAO) and polyaniline-grafted gelatine (NH2-Gel-PANI) was developed as a material in which to load neural stem cells (NSCs) and donepezil (DPL) to facilitate nerve regeneration after SCI. To evaluate the potential of the prepared NGP hydrogel in SCI repair applications, the surface morphology, self-repairing properties, electrical conductivity and cytocompatibility of the resulting hydrogel were analysed. Meanwhile, we evaluated the neural repair ability of NGP hydrogels loaded with DPL and NSCs using a rat model of spinal cord injury.ResultsThe NGP hydrogel has a suitable pore size, good biocompatibility, excellent conductivity, and injectable and self-repairing properties, and its degradation rate matches the repair cycle of spinal cord injury. In addition, DPL could be released continuously and slowly from the NGP hydrogel; thus, the NGP hydrogel could serve as an excellent carrier for drugs and cells. The results of in vitro cell experiments showed that the NGP hydrogel had good cytocompatibility and could significantly promote the neuronal differentiation and axon growth of NSCs, and loading the hydrogel with DPL could significantly enhance this effect. More importantly, the NGP hydrogel loaded with DPL showed a significant inhibitory effect on astrocytic differentiation of NSCs in vitro. Animal experiments showed that the combination of NGP hydrogel, DPL, and NSCs had the best therapeutic effect on the recovery of motor function and nerve conduction function in rats. NGP hydrogel loaded with NSCs and DPL not only significantly increased the myelin sheath area, number of new neurons and axon area but also minimized the area of the cystic cavity and glial scar and promoted neural circuit reconstruction.ConclusionsThe DPL- and NSC-laden electroactive hydrogel developed in this study is an ideal biomaterial for the treatment of traumatic spinal cord injury.

Melatonin rescues cerebral ischemic events through upregulated tunneling nanotube-mediated mitochondrial transfer and downregulated mitochondrial oxidative stress in rat brain

BackgroundCerebral ischemic events, comprising of excitotoxicity, reactive oxygen production, and inflammation, adversely impact the metabolic-redox circuit in highly active neuronal metabolic profile which maintains energy-dependent brain activities. Therefore, we investigated neuro-regenerative potential of melatonin (Mel), a natural biomaterial secreted by pineal gland. MethodsWe specifically determined whether Mel could influence tunneling nanotubes (TNTs)-mediated transfer of functional mitochondria (Mito) which in turn may alter membrane potential, oxidative stress and apoptotic factors. In vitro studies assessed the effects of Mito on levels of cytochrome C, mitochondrial transfer, reactive oxygen species, membrane potential and mass, which were all further enhanced by Mel pre-treatment, whereas in vivo studies examined brain infarct area (BIA), neurological function, inflammation, brain edema and integrity of neurons and myelin sheath in control, ischemia stroke (IS), IS + Mito and IS + Mel-Mito group rats. ResultsResults showed that Mel pre-treatment significantly increased mitochondrial transfer and antioxidants, and inhibited apoptosis. Mel-pretreated Mito also significantly reduced BIA with improved neurological function. Apoptotic, oxidative-stress, autophagic, mitochondrial/DNA-damaged biomarkers indices were also improved. ConclusionConclusively, Mel is a potent biomaterial which could potentially impart neurogenesis through repairing impaired metabolic-redox circuit via enhanced TNT-mediated mitochondrial transfer, anti-oxidation, and anti-apoptotic activities in ischemia.

Intraneural IFG-1 in Cryopreserved Nerve Isografts Increase Neural Regeneration and Functional Recovery in the Rat Sciatic Nerve.

Insulin-like growth factor 1 (IGF-1) was found to stimulate Schwann cell mitosis. Exogenous IGF-1 may improve nerve regeneration after cryopreservation. To evaulate the effect of intraneural administration of IGF-1 in cryopreserved nerve isografts. Eighteen millimeter grafts were used for bridging an 18-mm defect in the rat sciatic nerve. A total of 57 rats were randomly divided into three groups: (1) autograft (Group 1); (2) cryopreserved isograft (Group 2); (3) cryopreserved isograft with intraneural IGF-1 administration (Group 3). 12 weeks after surgery, functional recovery (Sciatic functional index [SFI], Swing speed [SS], nerve conduction velocity [NCV], amplitude of compound motor action potentials [CMAP], and gastrocnemius muscle index [GMI]) and nerve regeneration (myelin sheath area, total fiber counts, fiber density, and fiber width) were all evaluated. The intraneural injection of IGF-1 significantly improved SFI and SS at weeks 10 and 12. There were no statistical differences between Groups 1 and 3 in any of the SFI or SS evaluations. CMAP and NCV in Group 1 were significantly higher than in Groups 2 and 3, and Group 3 had significantly higher CMAP and NCV compared to Group 2. No significant differences were found in fiber width. The number of nerve fibers, percentage of myelinated fibers, fiber density, and GMI was significantly higher in Group 1 compared to Group 2, but no significant differences were found between Groups 1 and 3. The results show that intraneural injection of IGF-1 in an 18 mm cryopreserved isograft improve axonal regeneration and functional recovery.

Dose-dependent neurotoxicity caused by the addition of perineural dexmedetomidine to ropivacaine for continuous femoral nerve block in rabbits.

ObjectiveThis study was designed to evaluate the neurotoxicity of dexmedetomidine combined with ropivacaine for continuous femoral nerve block in rabbits.MethodsThirty New Zealand rabbits were randomly divided into 5 groups of 6 rabbits each and received a continuous femoral nerve block with saline; 0.25% ropivacaine; or 1, 2, or 3 µg/mL of dexmedetomidine added to 0.25% ropivacaine (Groups A–E, respectively). Sensory and motor function was assessed after the nerve block. The rabbits were anesthetized and killed after 48 hours of a continuous femoral nerve block, and the femoral nerves were removed for light and electron microscopy analyses.ResultsThe behavior scores were highest in Group A at 2 and 6 hours after injection. The scores were higher in Groups B and C than in Groups D and E at these same time points. All groups showed normal pathological tissues in the femoral nerves under optical microscopy. Under electron microscopy, histological abnormalities were observed only in Group E; none of the other groups exhibited pathological abnormalities. Quantitative analysis of the myelin sheath area revealed no significant difference in the axonal area, total area of the myelin sheath, or ratio of the total axonal area to the total area of the myelin sheath in all groups.ConclusionThe lowest doses of dexmedetomidine (1 and 2 µg/mL) combined with 0.25% ropivacaine for continuous femoral nerve block resulted in no neurotoxic lesions, but the higher dose (3 µg/mL) resulted in neurotoxic lesions in this rabbit experimental model.

Comparison of peripheral nerve protection between insulin-based glucose control and alpha lipoic acid (ALA) in the streptozotocin (STZ)-induced diabetic rat.

Strict glucose control is a well-proven therapeutic approach for peripheral neuropathies in patients with diabetes. Alpha lipoic acid (ALA) has also been accepted as a therapeutic agent for diabetic peripheral neuropathy (DPN) in the respect of pathogenesis. However, the potential of ALA as a treatment for DPN in comparison to that of glucose control is unclear. In this study, we compared the neuroprotective potential of glucose control and ALA. Animals were divided into 6 groups based on the intervention used, as follows: normal, diabetes (DM), DM+racemic form of ALA, DM+R form of ALA, DM+once daily insulin glargine, and DM+once daily insulin glargine with twice daily insulin glulisine. Various sensory tests were performed after 12 weeks of treatment, and immunohistochemistry of nerve fibers obtained from the sciatic and cutaneous nerves was performed after 24 weeks of treatment. There were no significant differences between the ALA-treated and insulin-treated DM groups in the sensory tests or in antioxidant activity. The axonal diameters and myelin sheath area of the sciatic nerves and the cutaneous small nerves, as assessed based on intraepidermal nerve fiber density, were similar in the ALA-treated and insulin-treated animals, although there was a non-significant trend for a mild increase in the both basal and rapid-acting insulin group compared with non-treated DM group. In conclusion, our results suggest that the neuroprotective benefits of ALA and insulin-based glucose control may be similar, although glucose control may have had slightly more beneficial effects in this animal model of diabetes. Of note, glucose levels should be strictly controlled, including corrections for fluctuations in the glucose level, to obtain therapeutic benefits in DPN.

Stimulation of morphofunctional repair of the facial nerve with photobiomodulation, using the end-to-side technique or a new heterologous fibrin sealant

This research evaluated the influence of Photobiomodulation Therapy (PBMT) on lesions of the facial nerve repaired with the end-to-side technique or coaptation with a new heterologous fibrin sealant. Thirty-two Wistar rats were separated into 5 groups: Control group (CG), where the buccal branch of the facial nerve was collected; Experimental Suture Group (ESG) and Experimental Fibrin Group (EFG), in which the buccal branch was end-to-side sutured to the zygomatic branch on the right side of the face or coaptated with fibrin sealant on the left side; Experimental Suture Laser Group (ESLG) and Experimental Fibrin Laser Group (EFLG), in which the same procedures were performed as the ESG and EFG, associated with PBMT (wavelength of 830nm, energy density 6.2J/cm2, power output 30mW, beam area of 0.116cm2, power density 0.26W/cm2, total energy per session 2.16J, cumulative dose of 34.56J). The laser was applied for 24s/site at 3 points on the skin's surface, for a total application time of 72s, performed immediately after surgery and 3 times a week for 5weeks. A statistically significant difference was observed in the fiber nerve area between the EFG and EFLG (57.49±3.13 and 62.52±3.56μm2, respectively). For the area of the axon, fiber diameter, axon diameter, myelin sheath area and myelin sheath thickness no statistically significant differences were found (p<0.05). The functional recovery of whisker movement occurred faster in the ESLG and EFLG, which were associated with PBMT, with results closer to the CG. Therefore, PBMT accelerated morphological and functional nerve repair in both techniques.

Endurance training induces structural and morphoquantitative changes in rat vagus nerve

ABSTRACT Introduction: Many nervous system tissues and cells suffers positive changes when faced to exercise training. However, data on vagus nerve adaptation from exercise-induced study is absent. Objective: To analyze the effect of an endurance training on the vagus nerve morphology of rats. Methods: Wistar rats (6 months of age) were divided into two groups: control group (CG, n=8), and aerobic trained group (AT, n=8). AT was submitted to a treadmill training program of five times per week during 12 weeks. The maximum speed stipulated in the training protocol corresponded to 60% of the mean maximum intensity achieved by the group in the test of maximum effort. Results: Twelve weeks of treadmill training resulted in left ventricular hypertrophy in the AT group com-pared to CG. There was a significant increase in the area of both the myelinated and unmyelinated axons, and in the area of myelin sheath with training. The number of neurotubules and neurofilaments in myelinated fibers of aerobic trained group was significantly greater than CG (p≤0.05). Conclusion: Endurance training promoted significant increase in morphometric parameters of the vagus nerve in the same way it affect somatic nerves.

Protective effect of mulberry flavonoids on sciatic nerve in alloxan-induced diabetic rats

Mulberry leaves (Morus alba L.) are a traditional Chinese medicine for blood serum glucose reduction. This study evaluated the protective effects of mulberry flavonoids on sciatic nerve in alloxan-induced diabetic rats. In this study, 80 Sprague-Dawley rats were divided into five groups: A (control), B (diabetic treated with saline), C-D (diabetic treated with 0.3, 0.1 g/kg mulberry flavonoids once a day for 8 weeks) and E (diabetic treated with 0.3 mg/kg methycobal). The diabetic condition was induced by intraperitoneal injection of 200 mg/kg alloxan dissolved in saline. At the end of the experimental period, blood, and tissue samples were obtained for biochemical and histopathological investigation. Treatment with 0.3 g/kg mulberry flavonoids significantly inhibited the elevated serum glucose (P&lt; 0.01). The increased myelin sheath area (P&lt; 0.01), myelinated fiber cross-sectional area and extramedullary fiber number (P&lt; 0.05) were also reduced in alloxan-induced rats treated with 0.3 g/kg mulberry flavonoids. 0.3 g/kg mulberry flavonoids also markedly decreased onion-bulb type myelin destruction and degenerative changes of mitochondria and Schwann cells. These findings demonstrate that mulberry flavonoids may improve the recovery of a severe peripheral nerve injury in alloxan-induced diabetic rats and is likely to be useful as a potential treatment on peripheral neuropathy (PN) in diabetic rats.

L-carnitine alleviates sciatic nerve crush injury in rats: functional and electron microscopy assessments.

Several studies have demonstrated that L-carnitine exhibits neuroprotective effects on injured sciatic nerve of rats with diabetes mellitus. It is hypothesized that L-carnitine exhibits neuroprotective effects on injured sciatic nerve of rats. Rat sciatic nerve was crush injured by a forceps and exhibited degenerative changes. After intragastric administration of 50 and 100 mg/kg L-carnitine for 30 days, axon area, myelin sheath area, axon diameter, myelin sheath diameter, and numerical density of the myelinated axons of injured sciatic nerve were similar to normal, and the function of injured sciatic nerve also improved significantly. These findings suggest that L-carnitine exhibits neuroprotective effects on sciatic nerve crush injury in rats.

Ultrastructural Morphometry of the Aortic Depressor Nerves and Extrinsic Renal Nerves: Similarities and Differences between Mice and Rats

Anatomical and physiological aspects involving rats and mice have shown similarities and differences between these experimental animal models. In cardiovascular physiology research, rats are being substituted by mice since mice are more susceptible to genetic manipulation. Nevertheless, little is known about mice normal anatomy and/or physiology to allow the correct interpretation of altered responses on genetically manipulated animals. We compared morphometric ultrastructural parameters of the aortic depressor nerve (ADN) and extrinsic renal nerve (SRN) between Wistar rats and C57BL/6J mice. After spontaneous activity recordings together with the arterial pressure pulse, ADN and SRN were dissected and prepared for light and transmission electron microscopy. Morphometry was performed with an image analysis software and took into consideration the fascicle area and diameter, myelinated and unmyelinated fiber number, density, area and diameter, myelin sheath area and the g-ratio. Comparisons were made for the same nerve between mice and rats and differences were considered significant when p<0.05. Both nerves were large in rats compared to mice, as were the myelinated fibers. On the other hand, unmyelinated fibers density, size and distributions were not different between species. These results suggest physiological differences on the fast conduction fibers between species, with possible different functional role. Morphological comparisons of the quantitative composition of peripheral nerves in different species and strains are very rare. Our study contributes to a morphological understanding of important nerves related to cardiovascular reflexes in two animal models. It also presents, for the first time, ultrastructural morphometric characteristics of the ADN in mice. This data provide a morphological basis for further studies involving functional investigations for reflex regulation of circulation in experimental model of hypertension and to improved cardiovascular system knowledge.

Reproducibility in Nerve Morphometry: Comparison between Methods and among Observers

We investigated the reproducibility of a semiautomated method (computerized with manual intervention) for nerve morphometry (counting and measuring myelinated fibers) between three observers with different levels of expertise and experience with the method. Comparisons between automatic (fully computerized) and semiautomated morphometric methods performed by the same computer software using the same nerve images were also performed. Sural nerves of normal adult rats were used. Automatic and semiautomated morphometry of the myelinated fibers were made through the computer software KS-400. Semiautomated morphometry was conducted by three independent observers on the same images, using the semiautomated method. Automatic morphometry overestimated the myelin sheath area, thus overestimating the myelinated fiber size and underestimating the axon size. Fiber distributions overestimation was of 0.5 μm. For the semiautomated morphometry, no differences were found between observers for myelinated fiber and axon size distributions. Overestimation of the myelin sheath size of normal fibers by the fully automatic method might have an impact when morphometry is used for diagnostic purposes. We suggest that not only semiautomated morphometry results can be compared between different centers in clinical trials but it can also be performed by more than one investigator in one single experiment, being a reliable and reproducible method.

Two nerve morphometry methods performed through the same computer program: are they comparable?

Morphometry has been widely used in studies designed to establish normal patterns of biological structures or to compare changes resulting from pathological processes. It can be performed manually or through semi‐automatic or automatic methods. However, it is difficult to compare results between studies that used different methods. We aimed to compare the results of two different methods of nerve morphometry, automatic and semi‐automatic, using the same computer software. Female Wistar rats (n = 5) with 90 days of age had their right and left sural nerves prepared for light microscopy. Automatic and semi‐automatic morphometry was performed using the program KS‐400 and took into consideration: area and lesser diameter of myelinated fibers and their axons, the myelin sheath area and the ratio between the lesser diameter of the axon and its respective myelinated fiber (G‐ratio). The nerves are longitudinally and laterally symmetric with both morphometric methods. A significant overestimation of the myelinated fibers size and a significant underestimation of the myelinated axons size were observed with the automatic method. Our results indicate an important differences in the values of the myelinated fibers morphometry of one nerve, obtained with two different methodologies. Thus, the same methodology must be standardized and used throughout a single experiment.Grant Funding Source : FAPESP, CNPq and FAEPA

Functional and morphometric differences between the early and delayed use of phototherapy in crushed median nerves of rats

This study evaluated the functional and quantitative differences between the early and delayed use of phototherapy in crushed median nerves. After a crush injury, low-level laser therapy (GaAs) was applied transcutaneously at the injury site, 3 min daily, with a frequency of five treatments per week for 2 weeks. In the early group, the first laser treatment started immediately after surgery, and in the delayed group, after 7 days. The grasping test was used for functional evaluation of the median nerve, before, 10, and 21 days after surgery, when the rats were killed. Three segments of the median nerve were analyzed histomorphometrically by light microscopy and computer analysis. The following features were observed: myelinated fiber and axon diameters, myelin sheath area, g-ratio, density and number of myelinated fibers, and area and number of capillaries. In the proximal segment (site of crush), the nerves of animals submitted to early and delayed treatment showed myelinated fiber diameter and myelin sheath area significantly larger compared to the untreated group. In the distal segment, the myelin sheath area was significantly smaller in the untreated animals compared to the delayed group. The untreated, early, and delayed groups presented a 50, 57, and 81% degree of functional recovery, respectively, at 21 days after injury, with a significant difference between the untreated and delayed groups. The results suggest that the nerves irradiated with low-power laser exhibit myelinated fibers of greater diameter and a better recovery of function.

Maturational changes in diffusion anisotropy in the rat corpus callosum: Comparison with quantitative histological evaluation

To determine the main histological components that affect fractional anisotropy (FA) in postnatal development of the rat corpus callosum and compare FA values with histological changes evaluated quantitatively. Diffusion tensor image (DTI) data of the rat (postnatal 1-10 weeks) corpus callosum were obtained with a 7.0 T MR scanner. Histological parameters were quantitatively assessed in toluidine blue-stained semithin sections. Simple and multiple linear regression analyses were performed to investigate relationships between FA values and histological variables. The mean FA value (mFA) increased significantly in the early growth stages, whereas the change became smaller after postnatal week 4. Simple regression analysis showed a high correlation between the area of myelin sheath and mFA (r = 0.856; P < 0.01). The area of extracellular space correlated negatively with mFA (r = -0.813; P < 0.01). In a forward stepwise analysis, the area of myelin sheath had the strongest influence on mFA (P < 0.001), followed by the number of unmyelinated axons (P = 0.113). Multiple linear regression analysis revealed that both parameters predicted mFA with a highly significant adjusted correlation coefficient (r(2) adj. = 0.738, P < 0.001). During the early development stage in the rat corpus callosum, the strongest contribution to FA value is the area of myelin sheath.

Relation between myelin area and axon diameter in the aortic depressor nerve of spontaneously hypertensive rats

The hypothesis that the aortic depressor nerve (ADN) from spontaneously hypertensive rats (SHR) does not show the expected correlation between myelin sheath area and the axonal diameter of myelinated fibers detected in normotensive rat myelinated fibers was tested by means of regression analysis. Proximal and distal segments of ADN from 13 normotensive Wistar–Kyoto rats (WKY) and nine SHR were prepared for light microscopy study. With an image analysis system, the area of the myelin sheath and the axonal diameter of all myelinated fibers in each nerve were automatically measured. Regression lines were calculated for all nerve segments from each group. Differences between the regression lines were tested for slope and intercept and differences between the correlation coefficients were also tested. Regression lines for WKY data showed no differences between the proximal and distal segments either for slope or intercept. Proximal and distal SHR regression lines were not coincident between segments or when compared to WKY data. These results agree with previous observations that there are morphological differences between WKY and SHR myelinated fibers of the ADN suggesting that the SHR depressor nerve fibers present characteristics of axonal atrophy and/or remyelination.

The end-to-side peripheral nerve repair: Functional and morphometric study using the peroneal nerve of rats

Morphologic and functional recovery following an end-to-side repair was studied comparatively with conventional end-to-end repair in a model of peroneal nerve lesion in rats. Twenty-eight rats were used and divided into four groups according to the reparative procedure following nerve division: (1) nerve stumps buried into neighboring muscles ( n=8); (2) conventional end-to-end repair ( n=7); (3) end-to-side repair onto the tibial nerve ( n=8); (4) sham operation ( n=5). The sciatic functional index (SFI) was evaluated at weekly intervals for 8 weeks, the peroneal nerve being resected on the 56th day for histologic and morphometric studies. The SFI progressively improved in Groups 2 (−16.9) and 3 (−22.7), although it did not reach normal values (around −8). The average nerve fiber density increased to normal values in both Groups 2 and 3, although accompanied by a marked decrease of average minimal and maximal nerve fiber diameter, myelin sheath area and G quotient. The differences between Groups 2 and 3 or Groups 2 and 4 were not significant. We conclude that, although resulting in significant morphologic and functional recovery, end-to-side repair is not as efficient as the conventional end-to-end nerve repair. However, end-to-side repair has a potential for application in selected cases in humans.

Effect of alpha-tocopherol supplementation on the ultrastructural abnormalities of peripheral nerves in experimental diabetes.

Ultrastructural observations were made on myelinated fibers in the tibial nerves in order to investigate the beneficial effects of alpha-tocopherol administration in streptozotocin-diabetic rats. Male Wistar rats, aged 12 weeks and weighing between 250 g to 300 g were studied. Six onset control rats were used to obtain the baseline parameters for this strain and age. Further 3 groups--untreated diabetic animals, diabetic animals treated with alpha-tocopherol, and age-matched controls--were studied over a 3-month period. In the diabetic animal, administration of alpha-tocopherol resulted in a significant increase (p < 0.05) in total plasma vitamin E levels when compared with other groups. Myelinated fiber cross-sectional area (p < 0.05), axonal area (p < 0.01) and myelin sheath area (p < 0.05) were significantly less in the tibial nerve of diabetic animals than in age-matched controls, but not different from those of onset controls. In the alpha-tocopherol treated diabetic animals, the values for these parameters were intermediate without showing significant difference when compared with age-matched controls and untreated diabetics. The "g" ratio (axon to fiber area) did not differ between any experimental groups. The number of large myelinated fibers were less in the untreated diabetic animals, but in the alpha-tocopherol-treated diabetics, the values were significantly higher (p < 0.05) than with untreated diabetics and were similar to those of age-matched controls. In conclusion, this ultrastructural study reiterated the fact that structural abnormalities of myelinated fibers occur in experimental diabetes and that alpha-tocopherol administration may be useful in preventing the development of these abnormalities.

Adenoviral gene therapy with catalase suppresses experimental optic neuritis.

To determine if adenoviral-mediated transfer of the gene for catalase (CAT), the reactive oxygen species scavenger, suppresses experimental optic neuritis. Gene therapy with CAT delivered by an adeno-associated viral vector was previously shown to suppress experimental optic neuritis. Because the transduction of protein expression with recombinant adeno-associated viral vector is relatively slow, taking weeks to reach full levels, we studied the effects of replication-deficient adenovirus containing CAT in suppressing experimental optic neuritis. Transduction with adenovirus occurs within days of inoculation, thus, it may be more applicable for the treatment of patients with acute optic neuritis. Replication-deficient adenovirus containing CAT was injected above the right optic nerve heads of SJL/J mice that were simultaneously sensitized for experimental allergic encephalomyelitis. For controls, the left eyes were injected with the replication-deficient adenovirus without CAT or no virus. The histological effects of CAT on the lesions of experimental allergic encephalomyelitis were measured by computerized analysis of the myelin sheath area (for demyelination), optic disc area (for optic nerve head swelling), the extent of the cellular infiltrate, extravasated serum albumin labeled with immunogold (for disruption of the blood-brain barrier), and the in vivo hydrogen peroxide reaction product. After 1 month, cell-specific catalase activity, evaluated by the quantitation of catalase immunogold, was increased about 2-fold each in endothelia, oligodendroglia, astrocytes, and axons of the CAT-inoculated right optic nerves compared with the control left optic nerves. The increased cellular levels of catalase reduced demyelination by 30%, optic nerve head swelling by 25%, cellular infiltration by 26%, disruption of the blood-brain barrier by 61%, and in vivo levels of hydrogen peroxide by 81%. Adenoviral-mediated gene transfer increased catalase levels in all optic nerve cell types, and it persisted for 1 month after inoculation. The increased cellular levels of catalase suppressed demyelination and blood-brain barrier disruption at the foci in the optic nerve where prior magnetic resonance imaging and histopathologic studies have demonstrated the demyelinating inflammation of experimental and human optic neuritis. Together, they suggest that gene therapy with CAT may be helpful in the treatment of patients with optic neuritis.

Adeno-associated viral-mediated catalase expression suppresses optic neuritis in experimental allergic encephalomyelitis.

Suppression of oxidative injury by viral-mediated transfer of the human catalase gene was tested in the optic nerves of animals with experimental allergic encephalomyelitis (EAE). EAE is an inflammatory autoimmune disorder of primary central nervous system demyelination that has been frequently used as an animal model for the human disease multiple sclerosis (MS). The optic nerve is a frequent site of involvement common to both EAE and MS. Recombinant adeno-associated virus containing the human gene for catalase was injected over the right optic nerve heads of SJL/J mice that were simultaneously sensitized for EAE. After 1 month, cell-specific catalase activity, evaluated by quantitation of catalase immunogold, was increased approximately 2-fold each in endothelia, oligodendroglia, astrocytes, and axons of the optic nerve. Effects of catalase on the histologic lesions of EAE were measured by computerized analysis of the myelin sheath area (for demyelination), optic disc area (for optic nerve head swelling), extent of the cellular infiltrate, extravasated serum albumin labeled by immunogold (for blood-brain barrier disruption), and in vivo H2O2 reaction product. Relative to control, contralateral optic nerves injected with the recombinant virus without a therapeutic gene, catalase gene inoculation reduced demyelination by 38%, optic nerve head swelling by 29%, cellular infiltration by 34%, disruption of the blood-brain barrier by 64%, and in vivo levels of H2O2 by 61%. Because the efficacy of potential treatments for MS are usually initially tested in the EAE animal model, this study suggests that catalase gene delivery by using viral vectors may be a therapeutic strategy for suppression of MS.