Bilateral Neuropathy of Primary Sensory Neurons by the Chronic Compression of Multiple Unilateral DRGs.

To mimic multilevel nerve root compression and intervertebral foramina stenosis in human, we established a new animal model of the chronic compression of unilateral multiple lumbar DRGs (mCCD) in the rat. A higher occurrence of signs of spontaneous pain behaviors, such as wet-dog shaking and spontaneous hind paw shrinking behaviors, was firstly observed from day 1 onward. In the meantime, the unilateral mCCD rat exhibited significant bilateral hind paw mechanical and cold allodynia and hyperalgesia, as well as a thermal preference to 30°C plate between 30 and 35°C. The expression of activating transcription factor 3 (ATF3) was significantly increased in the ipsilateral and contralateral all-sized DRG neurons after the mCCD. And the expression of CGRP was significantly increased in the ipsilateral and contralateral large- and medium-sized DRG neurons. ATF3 and CGRP expressions correlated to evoked pain hypersensitivities such as mechanical and cold allodynia on postoperative day 1. The results suggested that bilateral neuropathy of primary sensory neurons might contribute to bilateral hypersensitivity in the mCCD rat.

Dose-dependent protective effect of lithium chloride on retinal ganglion cells is interrelated with an upregulated intraretinal BDNF after optic nerve transection in adult rats.

Neuroprotection of lithium for axotomized retinal ganglion cells (RGCs) is attributed to upregulated intraretinal Bcl-2. As lithium also upregulates brain-derived neurotrophic factor (BDNF) which can rescue axotomized RGCs, it is hypothesized that lithium could protect RGCs through BDNF. This study investigated this hypothesis and a possible relationship between the dose and protection of lithium. All adult experimental rats received daily intraperitoneal injections of lithium chloride (LiCl) at 30, 60 or 85 mg/kg·bw until they were euthanized 2, 7 or 14 days after left intraorbital optic nerve (ON) transection. Our results revealed that RGC densities promoted and declined with increased dose of LiCl and the highest RGC densities were always in the 60 mg/kg·bw LiCl group at both 7 and 14 day points. Similar promotion and decline in the mRNA and protein levels of intraretinal BDNF were also found at the 14 day point, while such BDNF levels increased in the 30 mg/kg·bw LiCl group but peaked in the 60 and 85 mg/kg·bw LiCl groups at the 7 day point. These findings suggested that lithium can delay the death of axotomized RGCs in a dose-dependent manner within a certain period after ON injury and such beneficial effect is interrelated with an upregulated level of intraretinal BDNF.

Systemic injection of low-dose lipopolysaccharide fails to break down the blood-brain barrier or activate the TLR4-MyD88 pathway in neonatal rat brain.

We aimed to investigate whether peripheral low-dose lipopolysaccharide (LPS) induces the breakdown of the blood-brain barrier (BBB) and/or the activation of toll-like receptor 4 (TLR4) in the neonatal rat brain. Neonatal rats received intraperitoneal injections of low-dose LPS (0.3 mg/kg∙bw), and the BBB compromise was detected by Evans Blue extravasation and electron microscopy. Meanwhile, TLR4, adaptin myeloid differentiation factor 88 (MyD88), nuclear transcription factor kappa-B (NF-κB) p50 and tumor necrosis factor alpha (TNFα) in the neonatal rat brain were determined by quantitative real-time polymerase chain reaction (PCR) and Western Blot. Immunohistochemistry was used to determine the distribution and activation of microglia in the brain after LPS administration. It was demonstrated that Evans Blue extravasation was not observed in the brain parenchyma, and that tight junctions of cerebral endothelial cells remained intact after systemic injections of LPS in neonatal rats. Although intracerebroventricular injections of LPS activated microglia and up-regulated the expression of TLR4, MyD88, NF-κB p50 and TNFα in the neonatal rat brain, systemic LPS did not induce these responses. These findings indicate that while the neonatal rat brain responds to the direct intra-cerebral administration of LPS through robust TLR4 activation, systemic low-dose LPS does not induce the innate immune reaction or compromise the BBB in neonatal rats.

Restoring mitochondrial cardiolipin homeostasis reduces cell death and promotes recovery after spinal cord injury.

Alterations in phospholipids have long been associated with spinal cord injury (SCI). However, their specific roles and signaling cascades in mediating cell death and tissue repair remain unclear. Here we investigated whether alterations of cardiolipin (CL), a family of mitochondrion-specific phospholipids, play a crucial role in mitochondrial dysfunction and neuronal death following SCI. Lipidomic analysis was used to determine the profile of CL alteration in the adult rat spinal cord following a moderate contusive SCI at the 10th thoracic (T10) level. Cellular, molecular, and genetic assessments were performed to determine whether CL alterations mediate mitochondrial dysfunction and neuronal death after SCI, and, if so, whether reversing CL alteration leads to neuroprotection after SCI. Using lipidomic analysis, we uncovered CL alterations at an early stage of SCI. Over 50 distinct CL species were identified, of which 50% showed significantly decreased abundance after SCI. The decreased CL species contained mainly polyunsaturated fatty acids that are highly susceptible to peroxidation. In parallel, 4-HNE, a lipid peroxidation marker, significantly increased after SCI. We found that mitochondrial oxidative stress not only induced CL oxidation, but also resulted in CL loss by activating cPLA2 to hydrolyze CL. CL alterations induced mitochondrial dysfunction and neuronal death. Remarkably, pharmacologic inhibition of CL alterations with XJB-5-131, a novel mitochondria-targeted electron and reactive oxygen species scavenger, reduced cell death, tissue damage and ameliorated motor deficits after SCI in adult rats. These findings suggest that CL alteration could be a novel mechanism that mediates injury-induced neuronal death, and a potential therapeutic target for ameliorating secondary SCI.

Neutralization of BDNF attenuates the in vitro protective effects of olfactory ensheathing cell-conditioned medium on scratch-insulted retinal ganglion cells.

Transplantation of olfactory ensheathing cells (OECs) becomes one of the promising strategies in restoring lost functions of injured central nervous system. Elevated level of expressed brain-derived neurotrophic factor (BDNF) was revealed in the previous studies to be related to the protective effects of OECs on injured cortical and brain stem neurons as well as retinal ganglion cells (RGCs), but no evidence has been obtained to demonstrate whether transplanted OECs protect injured central neurons directly by their secreted BDNF. In the present study, the effects of BDNF neutralization on the neuroprotection of adult OEC-conditioned medium (OEC-CM) on scratch-insulted RGCs were examined. The results showed that OEC-CM protected cultured RGCs from scratch insult, and neutralization of BDNF by BDNF neutralizing antibody attenuated such neuroprotection of the medium. It is thus concluded that neurotrophic factors including BDNF secreted by OECs can protect injured OECs in vitro and BDNF plays a major role in such a protection of OECs.

Thymosin-beta4 attenuates ethanol-induced neurotoxicity in cultured cerebral cortical astrocytes by inhibiting apoptosis.

Thymosin-beta4 (Tbeta4) is a major actin monomer-binding peptide in mammalian tissues and plays a crucial role in the nervous system in synaptogenesis, neuronal survival and migration, axonal growth, and plastic changes of dendritic spines. However, it is unknown whether Tbeta4 is also involved in challenges with external stress such as ethanol-induced neurotoxicity. In the present study, we investigated the effects of Tbeta4 on ethanol-induced neurotoxicity in cultured cerebral cortical astrocytes and the underlying mechanisms. Primarily cultured astrocytes were treated with 1 microg/ml Tbeta4 2 h prior to administration of 100 mM ethanol for 0.5, 1, 3 and 6 days, respectively. The results showed that ethanol caused neurotoxicity in cultured astrocytes, as shown by declined cell viability, distinct astroglial apoptosis and increased intracellular peroxidation. Tbeta4 markedly promoted cell viability, ameliorated the injury of intracellular glial fibrillary acidic protein-immunopositive cytoskeletal structures, reduced the percentage of apoptotic astrocyte and cellular DNA fragmentation, suppressed caspase-3 activity and upregulated Bcl-2 expression, inhibited the accumulation of reactive oxygen species and production of malondialdehyde in ethanol-treated astrocytes in a time-dependent manner. These data indicated that Tbeta4 attenuates ethanol-induced neurotoxicity in cultured cortical astrocytes through inhibition of apoptosis signaling, and one of the mechanisms underlying the capacity of Tbeta4 to suppress apoptosis may in part be due to its effect of anti-peroxidation.

Evidence for heterogeneity of astrocyte de-differentiation in vitro: astrocytes transform into intermediate precursor cells following induction of ACM from scratch-insulted astrocytes.

Our previous study definitely demonstrated that the mature astrocytes could undergo a de-differentiation process and further transform into pluripotential neural stem cells (NSCs), which might well arise from the effect of diffusible factors released from scratch-insulted astrocytes. However, these neurospheres passaged from one neurosphere-derived from de-differentiated astrocytes possessed a completely distinct characteristic in the differentiation behavior, namely heterogeneity of differentiation. The heterogeneity in cell differentiation has become a crucial but elusive issue. In this study, we show that purified astrocytes could de-differentiate into intermediate precursor cells (IPCs) with addition of scratch-insulted astrocyte-conditioned medium (ACM) to the culture, which can express NG2 and A2B5, the IPCs markers. Apart from the number of NG2(+) and A2B5(+) cells, the percentage of proliferative cells as labeled with BrdU progressively increased with prolonged culture period ranging from 1 to 10 days. Meanwhile, the protein level of A2B5 in cells also increased significantly. These results revealed that not all astrocytes could de-differentiate fully into NSCs directly when induced by ACM, rather they generated intermediate or more restricted precursor cells that might undergo progressive de-differentiation to generate NSCs.

Long-term primary culture of highly-pure rat embryonic hippocampal neurons of low-density.

In order to develop a simplified method for long-term primary culture of highly-pure rat embryonic hippocampal neurons of low-density (10(3) cells/cm(2)), we optimized and modified conventional culturing methods. The modifications of our simplified method include: (1) combinational application of two growth substrates, tail collagen and poly-L-lysine, to coat plastic culture dishes and coverslips for a better neuronal attachment; (2) dissociation of hippocampal tissues with combinational use of two milder enzymes (collagenase and dispase) and trypsin of a lower concentration to minimize enzymatic damages to cultured neurons; (3) a cell pre-plating step to preliminarily eliminate the contaminating non-neuronal cells; (4) a modified culture medium as a critical step to promote highly pure neurons of low-density for a long term; and (5) appropriately reduced frequency and volume of refreshment of the culture medium. Using our modified method, the beta-tubulin III-immunostained and Hoechst 33342 counterstained neurons harvested a steady and healthy growth with a longer culture time of over 35 days, and a clear distinction between TAU-1- and MAP2-immunoreactive neurites was apparent at the early culturing period. In addition, the purity of neurons was over 95% at the different time points in comparison with the control culture using conventional serum-free method in which most neurons degenerated and died within 5 days. Thus, our modified method proved to be a simple, feasible as well as time- and resource-saving approach for a long-term survival of pure rat embryonic hippocampal neurons of low-density.

[The neuroglobin expression when retinal ganglion cells death in acute rats' retina ischemia].

OBJECTIVE: To investigate the intra-retinal expression of neuroglobin (Ngb) and death of retinal ganglion cells (RGCs) in acute retina ischemia rats. METHODS: It was an experimental study. The acute retina ischemia model was established by specific hypothesised left retina artery of Sprague-Dawley rats. Forty rats were divided into four groups (0, 15, 30, 60 min) by the time of retina ischemia. Every group has 10 rats, in one group random 3 rats were detected by Western blotting; 4 rats were detected by ganglion cell counted by hematoxylin and eosin stain and immunohistochemistry fluorescence intensity analysis. The rest 3 rats were detected by Western blotting. The difference among different data were analyzed statistically by One-factor analysis of variance and LSD-t analysis. RESULTS: The intra-retinal expression of Ngb reached maximum after acute ischemia 15 minute (P = 0.000). then the expression began decreasing. After 30 minute acute ischemia, the expression of Ngb had approached normal (P = 0.728), while, the cell number of RGCs began lower than 0 min group (P = 0.011); after 60 minute acute ischemia, the expression of Ngb had been obviously lower than 0 min group (P = 0.001), the cell number of RGCs had been further lower than 0 min group (P = 0.000). The expression of Ngb in RGCs layer was highest in rat retina. The expression in inner plexiform layer and external plexiform layer were lower than the former. The expression of Ngb RGCs was mostly intracytoplasm. After 30 minute acute ischemia, the expression of Ngb were detected in mitochondrial outer compartment and mitochondrial cristae, but in cytoplasm of inner nuclear layer and outer nuclear layer the Ngb was not found. CONCLUSION: Ngb quickly steps-up when RGCs die in acute retina ischemia, and mainly expresses intracytoplasm of RGCs. It has tense relationships with nerve cells' survival in hypoxia.

Etomidate affects the anti-oxidant pathway to protect retinal ganglion cells after optic nerve transection.

Our previous studies revealed that etomidate, a non-barbiturate intravenous anesthetic agent, has protective effects on retinal ganglion cells within 7 days after optic nerve transection. Whether this process is related to anti-oxidative stress is not clear. To reveal its mechanism, we established the optic nerve transection injury model by transecting 1 mm behind the left eyeball of adult male Sprague-Dawley rats. The rats received an intraperitoneal injection of etomidate (4 mg/kg) once per day for 7 days. The results showed that etomidate significantly enhanced the number of retinal ganglion cells retrogradely labeled with Fluorogold at 7 days after optic nerve transection. Etomidate also significantly reduced the levels of nitric oxide and malonaldehyde in the retina and increased the level of glutathione at 12 hours after optic nerve transection. Thus, etomidate can protect retinal ganglion cells after optic nerve transection in adult rats by activating an anti-oxidative stress response. The study was approved by the Animal Ethics Committee at Air Force Medical University, China (approval No. 20180305) on March 5, 2018.

Moderate prenatal alcohol exposure suppresses the TLR4-mediated innate immune response in the hippocampus of young rats.

Prenatal alcohol exposure (PAE) could lead to developmental disorders of the central nervous system (CNS) and mental retardation. Toll-like receptor (TLR) 4 plays an important role in PAE-induced neurodevelopmental defects. However, how PAE affects TLR4 response in the brain remains controversial. Using a moderate PAE model by feeding pregnant rats with liquid ethanol diet, we investigated the TLR4-mediated response to intraventricular injection of lipopolysaccharide (LPS) in the hippocampus of PEA rats at postnatal day (PND) 30. The results showed that PAE significantly up-regulated the expression of Toll-Interleukin-1 Receptor (TIR)-domain-containing adaptor protein inducing interferon (IFN)-ß (TRIF), TNF-α, and IL-1ß in the rat hippocampus in the absence of LPS, indicated by western blot assay. LPS treatment dramatically up-regulated the expressions of TLR4 and its downstream molecules in the hippocampus of paired-food and control groups. But no such significant changes of those molecules were found in the hippocampus of PAE animals. Moreover, the LPS stimulation even down-regulated the levels of TLR4 and TRIF in the PAE group. These data suggest that the relatively moderate level of PAE may lead to a mild neuroinflammation and a suppression of TLR4-mediated response to LPS in the hippocampus of young rats. As innate immunity plays crucial roles in CNS development, moderate PAE-induced suppression of TLR4-mediated response may serve as a new candidate mechanism of CNS developmental defects.

Branched-Chain Amino Acids Enhance Retinal Ganglion Cell Survival and Axon Regeneration after Optic Nerve Transection in Rats.

PURPOSE: This study's aim was to investigate the beneficial effects of branched-chain amino acids (BCAAs) on the neuronal survival and axon regeneration of retinal ganglion cells (RGCs) after optic nerve (ON) transection. METHOD: The experimental rats received daily BCAA injections through the caudal vein after left intra-orbital ON transection. Neuroprotection was evaluated by counting Fluorogold-labeled RGCs. The role of mammalian target of rapamycin (mTOR) pathway activation in promoting RGC survival was studied after rapamycin administration. Moreover, a peripheral nerve (PN) graft was transplanted onto the transected ON to study the effects of BCAAs on axon regeneration of injured RGCs. RESULTS: Our results showed that BCAAs alleviated the death of RGCs 7 and 14 days after ON transection, accompanied by an activation of mTOR pathway in RGCs. Blocking mTOR pathway with rapamycin eliminated such neuroprotective effects of BCAAs. Moreover, BCAAs also promoted axon regeneration of injured RGCs into a PN graft. CONCLUSION: Our results suggest a neuroprotection of BCAAs through the activation of mTOR pathway. BCAAs also have a beneficial effect on axon regeneration of injured RGCs. Therefore, BCAAs could be considered for the clinical treatment of ON injury.

Clinical Cell Therapy Guidelines for Neurorestoration (IANR/CANR 2017).

Cell therapy has been shown to be a key clinical therapeutic option for central nervous system diseases or damage. Standardization of clinical cell therapy procedures is an important task for professional associations devoted to cell therapy. The Chinese Branch of the International Association of Neurorestoratology (IANR) completed the first set of guidelines governing the clinical application of neurorestoration in 2011. The IANR and the Chinese Association of Neurorestoratology (CANR) collaborated to propose the current version "Clinical Cell Therapy Guidelines for Neurorestoration (IANR/CANR 2017)". The IANR council board members and CANR committee members approved this proposal on September 1, 2016, and recommend it to clinical practitioners of cellular therapy. These guidelines include items of cell type nomenclature, cell quality control, minimal suggested cell doses, patient-informed consent, indications for undergoing cell therapy, contraindications for undergoing cell therapy, documentation of procedure and therapy, safety evaluation, efficacy evaluation, policy of repeated treatments, do not charge patients for unproven therapies, basic principles of cell therapy, and publishing responsibility.

Nogo-66 receptor at the gap junctions between pituicytes of the rat.

Nogo-66 receptor was first identified in neurons. Recently, it was demonstrated in glial cells as well. Our previous study on the expression of Nogo-66 receptor in the cerebellum of the rat surprisingly found its location at the glial gap junctions. Here, we present our study on Nogo-66 receptor in the rat posterior pituitary, which is densely packed with pituicytes, a special type of astrocyte, and is known to be rich in gap junctions. We were able to demonstrate with immunohistochemistry and immuno-electron microscopy abundant Nogo-66 receptor immunoreactive gap junctions between pituicytes. This study, together with our prior one, strongly suggests that the Nogo-66 receptor may play a role in regulating the function of the gap junctions.

Large-scale reconstitution of a retina-to-brain pathway in adult rats using gene therapy and bridging grafts: An anatomical and behavioral analysis.

Peripheral nerve (PN) grafts can be used to bridge tissue defects in the CNS. Using a PN-to-optic nerve (ON) graft model, we combined gene therapy with pharmacotherapy to promote the long-distance regeneration of injured adult retinal ganglion cells (RGCs). Autologous sciatic nerve was sutured onto the transected ON and the distal end immediately inserted into contralateral superior colliculus (SC). Control rats received intraocular injections of saline or adeno-associated virus (AAV) encoding GFP. In experimental groups, three bi-cistronic AAV vectors encoding ciliary neurotrophic factor (CNTF) were injected into different regions of the grafted eye. Each vector encoded a different fluorescent reporter to assess retinotopic order in the regenerate projection. To encourage sprouting/synaptogenesis, after 6 weeks some AAV-CNTF injected rats received an intravitreal injection of recombinant brain-derived neurotrophic factor (rBDNF) or AAV-BDNF. Four months after surgery, cholera toxin B was used to visualize regenerate RGC axons. RGC viability and axonal regrowth into SC were significantly greater in AAV-CNTF groups. In some cases, near the insertion site, regenerate axonal density resembled retinal terminal densities seen in normal SC. Complex arbors were seen in superficial but not deep SC layers and many terminals were immunopositive for presynaptic proteins vGlut2 and SV2. There was improvement in visual function via the grafted eye with significantly greater pupillary constriction in both AAV-CNTF+BDNF groups. In both control and AAV-CNTF+rBDNF groups the extent of light avoidance correlated with the maximal distance of axonal penetration into superficial SC. Despite the robust regrowth of RGC axons back into the SC, axons originating from different parts of the retina were intermixed at the PN graft/host SC interface, indicating that there remained a lack of order in this extensive regenerate projection.

FTY720 enhances osteogenic differentiation of bone marrow mesenchymal stem cells in ovariectomized rats.

Sphingosine-1-phosphate and its structural analog FTY720 (fingolimod) are important in the inhibition of osteoclast differentiation and bone resorption, however, it remains unknown whether they enhance osteogenic differentiation of the bone marrow mesenchymal stem cells (BM­MSCs). The present study investigated the effect of FTY720 on the osteogenic differentiation of BM­MSCs from the femurs of the ovariectomized (OVX) rats. Three different concentrations (1, 10 and 100 nM) of FTY720 were demonstrated to markedly upregulate mRNA expression levels of Runt­related transcription factor 2 (Runx2) and Sp7 transcription factor (Sp7) at 2 weeks, and alkaline phosphatase (ALP) at 3 weeks. The osteocalcin (OCN) expression was similar at weeks 2 and 3. The protein expression levels of Runx2, Sp7, OCN and ALP induced by three different concentrations of FTY720 were higher than those in the control groups at 3 weeks in the OVX and sham groups. The findings of the current study suggested a beneficial effect of FTY720 on bone formation in OVX rats, and provided a potential therapeutic method of FTY720 to prevent alveolar bone resorption in patients with post­menopausal osteoporosis.

CNTF and BDNF have similar effects on retinal ganglion cell survival but differential effects on nitric oxide synthase expression soon after optic nerve injury.

PURPOSE: To investigate the effect of ciliary neurotrophic factor (CNTF) and brain-derived neurotrophic factor (BDNF) on retinal ganglion cell (RGC) survival and nitric oxide synthase (NOS) expression in the retina during the early phase of optic nerve (ON) injury, and to examine whether intraperitoneal application of the NOS scavenger nitro-l-arginine (l-NA) could protect the injured RGCs. METHODS: RGCs were retrogradely labeled with granular blue 3 days before the ON was intraorbitally transected. RGC survival was examined 1 week after ON transection and intraocular injection of CNTF and/or BDNF, or 1 to 2 weeks after daily intraperitoneal injection of the NOS inhibitor l-NA. NOS expression was examined by NADPH-diaphorase histochemistry and neuronal NOS (nNOS) immunohistochemistry, and nNOS-positive cells were identified by various staining approaches. RESULTS: Both CNTF and BDNF significantly increased RGC survival 1 week after ON injury. In the ganglion cell layer (GCL), CNTF did not increase the number of NADPH-diaphorase positive ((+)) cells but appeared to reduce the intensity of NADPH-diaphorase staining, whereas BDNF increased the number of NADPH-diaphorase(+) cells and also appeared to enhance the intensity of NADPH-diaphorase staining. In the GCL, amacrine cells but not RGCs were nNOS(+). Some macrophages were also nNOS(+). In contrast, no amacrine cells were nNOS(+) in the inner nuclear layer. Daily intraperitoneal injection of l-NA at appropriate concentrations promoted RGC survival for 1 or 2 weeks after ON injury. CONCLUSIONS: Both CNTF and BDNF protected RGCs after ON injury. CNTF and BDNF acted differently on NOS expression in the GCL. Intraperitoneal injections of l-NA at appropriate dosages enhance RGC survival.

[The role of cyclic AMP in repair of hemisection of spinal cord in rats models].

OBJECTIVE: To study the role of cAMP in repair of hemisection of spinal cord in rats models. METHODS: Rats models of spinal cord hemisection were made and cAMP were injected once in the motor cortex or continuously input in the lesion area or in the subarachnoid cistern for 3 d. NFs, GFAP, CSTs and spinal axons in the lesion areas were observed by immunohistochemistry and hind limb movements were evaluated in BBB scales. RESULTS: Many regenerated axons were presented in the lesion areas in cAMP groups though no continuous long regenerated axons traversed the lesion area when cAMP was input in the motor cortex or in the local lesion area. In control group, no regenerated axon were presented in the lesion areas. When cAMP was input in the subarachnoid cistern, only few-labelled CST axon survived and presented in the lesion area comparing no labelled CST axon presented in the lesion area. More NFs and less GFAP were distributed and extended in the lesion area in the cAMP groups. All the rats restored to normally walk 4-5 weeks after operations and no significance existed between cAMP groups and control groups comparing the BBB scales of hind limb movements. CONCLUSION: cAMP injected in the brain cortex or continuously input in the lesion area can induce the axonal regeneration.

Neuroprotective effect of inosine on axotomized retinal ganglion cells in adult rats.

PURPOSE: To explore the potential survival-promoting effect of inosine on axotomized retinal ganglion cells (RGCs) of adult rats in vivo. METHODS: The left optic nerves (ON) in the subject rats were transected at 1.5 mm from the optic disc. Repeated intraperitoneal injections or single intraocular injection of inosine were administered. The RGCs were retrogradely labeled with a gold fluorescent dye and the density of surviving RGCs in number per square millimeter of retina was calculated in wholemounted retinas. The functional integrity of the blood-retinal barrier (BRB) after ON transection was evaluated with an intravenous injection of Evans blue. RESULTS: In control animals, the mean density of surviving RGCs (number per square millimeter) of the whole retina was 2007 +/- 68 at 2 days (taken as the normal value), 927 +/- 156 at 7 days, and 384 +/- 33 at 14 days after surgery. Repeated intraperitoneal injections (75 mg/kg for each injection) of inosine significantly enhanced RGC survival at 14 days after ON transection (500 +/- 38), whereas no significant difference in the densities was detected at 7 days (974 +/- 101), even when the dosage of inosine was doubled (1039 +/- 61). At this time point, however, a single intraocular injection of inosine significantly increased the density of surviving RGCs (1184 +/- 156). Moreover, more RGCs around the optic disc were rescued when inosine, administered either intraperitoneally or intraocularly, showed a beneficial effect on RGC survival. No breakdown of the BRB after ON transection was detected with the method used in the study. CONCLUSIONS: These findings demonstrate that inosine could protect axotomized RGCs in vivo after ON transection.