Neurovascular dynamics of repeated cortical spreading depolarizations after acute brain injury.

Cortical spreading depolarizations (CSDs) are increasingly suspected to play an exacerbating role in a range of acute brain injuries, including stroke, possibly through their interactions with cortical blood flow. We use simultaneous wide-field imaging of neural activity and hemodynamics in Thy1-GCaMP6f mice to explore the neurovascular dynamics of CSDs during and following Rose Bengal-mediated photothrombosis. CSDs are observed in all mice as slow-moving waves of GCaMP fluorescence extending far beyond the photothrombotic area. Initial CSDs are accompanied by profound vasoconstriction and leave residual oligemia and ischemia in their wake. Later, CSDs evoke variable responses, from constriction to biphasic to vasodilation. However, CSD-evoked vasoconstriction is found to be more likely during rapid, high-amplitude CSDs in regions with stronger oligemia and ischemia, which, in turn, worsens after each repeated CSD. This feedback loop may explain the variable but potentially devastating effects of CSDs in the context of acute brain injury.

The Gliopeptide ODN, a Ligand for the Benzodiazepine Site of GABAA Receptors, Boosts Functional Recovery after Stroke.

Following stroke, the survival of neurons and their ability to reestablish connections is critical to functional recovery. This is strongly influenced by the balance between neuronal excitation and inhibition. In the acute phase of experimental stroke, lethal hyperexcitability can be attenuated by positive allosteric modulation of GABAA receptors (GABAARs). Conversely, in the late phase, negative allosteric modulation of GABAAR can correct the suboptimal excitability and improves both sensory and motor recovery. Here, we hypothesized that octadecaneuropeptide (ODN), an endogenous allosteric modulator of the GABAAR synthesized by astrocytes, influences the outcome of ischemic brain tissue and subsequent functional recovery. We show that ODN boosts the excitability of cortical neurons, which makes it deleterious in the acute phase of stroke. However, if delivered after day 3, ODN is safe and improves motor recovery over the following month in two different paradigms of experimental stroke in mice. Furthermore, we bring evidence that, during the subacute period after stroke, the repairing cortex can be treated with ODN by means of a single hydrogel deposit into the stroke cavity.SIGNIFICANCE STATEMENT Stroke remains a devastating clinical challenge because there is no efficient therapy to either minimize neuronal death with neuroprotective drugs or to enhance spontaneous recovery with neurorepair drugs. Around the brain damage, the peri-infarct cortex can be viewed as a reservoir of plasticity. However, the potential of wiring new circuits in these areas is restrained by a chronic excess of GABAergic inhibition. Here we show that an astrocyte-derived peptide, can be used as a delayed treatment, to safely correct cortical excitability and facilitate sensorimotor recovery after stroke.

Preventive Effects of Neuroprotective Agents in a Neonatal Rat of Photothrombotic Stroke Model.

Neonatal ischemic stroke has a higher incidence than childhood stroke. Seizures are the first sign for the need for clinical assessment in neonates, but many questions remain regarding treatments and follow-up modalities. In the absence of a known pathophysiological mechanism, only supportive care is currently provided. Stroke-induced microglia activation and neuroinflammation are believed to play a central role in the pathological progression of neonatal ischemic stroke. We induced a photothrombotic infarction with Rose Bengal in neonatal rats to investigate the effects of pre- and post-treatment with Aspirin (ASA), Clopidogrel (Clop), and Coenzyme Q10 (CoQ10), which are known for their neuroprotective effects in adult stroke. Pre-stroke medication ameliorates cerebral ischemic injury and reduces infarct volume by reducing microglia activation, cellular reactive oxygen species (ROS) production, and cytokine release. Post-stroke administration of ASA, Clop, and CoQ10 increased motor function and reduced the volume of infarction, and the statistical evidence was stronger than that seen in the pre-stroke treatment. In this study, we demonstrated that ASA, Clop, and CoQ10 treatment before and after the stroke reduced the scope of stroke lesions and increased behavioral activity. It suggests that ASA, Clop, and CoQ10 medication could significantly have neuroprotective effects in the neonates who have suffered strokes.

Gemcitabine loaded microbubbles for targeted chemo-sonodynamic therapy of pancreatic cancer.

Pancreatic cancer remains one of the most lethal forms of cancer with a 10-year survival of <1%. With little improvement in survival rates observed in the past 40 years, there is a significant need for new treatments or more effective strategies to deliver existing treatments. The antimetabolite gemcitabine (Gem) is the most widely used form of chemotherapy for pancreatic cancer treatment, but is known to produce significant side effects when administered systemically. We have previously demonstrated the benefit of combined chemo-sonodynamic therapy (SDT), delivered using oxygen carrying microbubbles (O2MB), as a targeted treatment for pancreatic cancer in a murine model of the disease. In this manuscript, we report the preparation of a biotin functionalised Gem ligand for attachment to O2MBs (O2MB-Gem). We demonstrate the effectiveness of chemo-sonodynamic therapy following ultrasound-targeted-microbubble-destruction (UTMD) of the O2MB-Gem and a Rose Bengal loaded O2MB (O2MB-RB) as a targeted treatment for pancreatic cancer. Specifically, UTMD using the O2MB-Gem and O2MB-RB conjugates reduced the viability of MIA PaCa-2, PANC-1, BxPC3 and T110299 pancreatic cancer cells by >60% (p < 0.001) and provided significant tumour growth delay (>80%, p < 0.001) compared to untreated animals when human xenograft MIA PaCa-2 tumours were treated in SCID mice. The toxicity of the O2MB-Gem conjugate was also determined in healthy non-tumour bearing MF1 mice and revealed no evidence of renal or hepatic damage. Therefore, the results presented in this manuscript suggest that chemo-sonodynamic therapy using the O2MB-Gem and O2MB-RB conjugates, is potentially an effective targeted and safe treatment modality for pancreatic cancer.

Rose Bengal attached and dextran coated gadolinium oxide nanoparticles for potential diagnostic imaging applications.

We report here, reverse micelle mediated synthesis of multifunctional dextran (dex) coated Gd2O3 nanoparticles (NPs) carrying rose bengal (RB) dye for magnetic resonance and optical imaging. The diameter of these RB attached dex coated Gd2O3 NPs (Gd-dex-RB NPs) was found to be ~17 nm as measured by TEM. NMR line broadening effect on the surrounding water protons affirmed the paramagnetic nature of these NPs. Optical properties of Gd-dex-RB NPs were validated by UV-Vis and fluorescence spectroscopy. Time dependent release profile of RB from NPs at two different pH of 7.4 and 5.0 revealed that these NPs behave as slow releasing system. In-vitro study revealed that NPs are efficiently taken up by cells and show optical activity in cellular environment. In vitro cell viability (SRB) assay was performed on cancerous (A-549, U-87) and normal (HEK-293) cell lines, showed the absence of cytotoxic effect of Gd-dex-RB NPs. Therefore, such multifunctional NPs can be efficiently used for bio-imaging and optical tracking.

Molecular imprinted chitosan-TiO2 nanocomposite for the selective removal of Rose Bengal from wastewater.

A new molecularly imprinted polymer (MIP) chitosan-TiO2 nanocomposite (CTNC) was prepared for the selective and quantitative removal of Rose Bengal dye from industrial wastewater. The physicochemical features of the prepared CTNC-MIP nanoparticles were thoroughly investigated. The prepared MIP nanoparticles exhibited high surface area (95.38m2/g) with relatively uniform mesoporous channels that allowed an exceptional uptake of the dye (qm=79.365mg/g) and reflected the high selectivity of the prepared MIP compared to pure chitosan. The dye uptake was investigated using Freundlich, Langmuir, Dubinin Radushkevich and Temkin models. The kinetics of removal was explored by pseudo-first, pseudo second order, Elovich and Weber-Morris models. The Experimental data fitted well into pseudo-second order model, and much well with the Langmuir isotherm confirming the formation of monolayer of dye molecules. The enthalpy of adsorption was (62.279kJmol-1) showing strong interaction of MIP with the dye. The prepared MIP exhibited a good recyclability and stability.

Zebrafish model of photochemical thrombosis for translational research and thrombolytic screening in vivo.

Acute thromboembolic diseases remain the major global cause of death or disability. Although an array of thrombolytic and antithrombotic drugs has been approved to treat or prevent thromboembolic diseases, many more drugs that target specific clotting mechanisms are under development. Here a novel zebrafish model of photochemical thrombosis is reported and its prospective application for the screening and preclinical testing of thrombolytic agents in vivo is demonstrated. Through photochemical excitation, a thrombus was induced to form at a selected section of the dorsal aorta of larval zebrafish, which had been injected with photosensitizers. Such photochemical thrombosis can be consistently controlled to occlude partially or completely the targeted blood vessel. Detailed mechanistic tests indicate that the zebrafish model of photochemical thrombosis exhibits essential features of classical coagulation and a thrombolytic pathway. For demonstration, tissue plasminogen activator (tPA), a clinically feasible thrombolytic agent, was shown to effectively dissolve photochemically induced blood clots. In light of the numerous unique advantages of zebrafish as a model organism, our approach is expected to benefit not only the development of novel thrombolytic and antithrombotic strategies but also the fundamental or translational research targeting hereditary thrombotic or coagulation disorders.

Photosensitized rose Bengal-induced phototoxicity on human melanoma cell line under natural sunlight exposure.

Rose Bengal (RB) is an anionic water-soluble xanthene dye, which used for many years to assess eye cornea and conjunctiva damage. RB showed strong absorption maxima (λmax) under visible light followed by UV-B and UV-A. RB under sunlight exposure showed a time-dependent photodegradation. Our results show that photosensitized RB generates (1)O2 via Type-II photodynamic pathway and induced DNA damage under sunlight/UV-R exposure. 2'dGuO degradation, micronuclei formation, and single- and double-strand breakage were the outcome of photogenotoxicity caused by RB. Quenching studies with NaN3 advocate the involvement of (1)O2 in RB photogenotoxicity. RB induced linoleic acid photoperoxidation, which was parallel to (1)O2-mediated DNA damage. Oxidative stress in A375 cell line (human melanoma cell line) was detected through DCF-DA assay. Photosensitized RB decreased maximum cellular viability under sunlight followed by UV-B and UV-A exposures. Apoptosis was detected as a pattern of cell death through the increased of caspase-3 activity, decreased mitochondrial membrane potential, and PS translocation through inner to outer plasma membrane. Increased cytosolic levels of Bax also advocate the apoptotic cell death. We propose a p53-mediated apoptosis via increased expression of Bax gene and protein. Thus, the exact mechanism behind RB phototoxicity was the involvement of (1)O2, which induced oxidative stress-mediated DNA and membrane damage, finally apoptotic cell death under natural sunlight exposure. The study suggests that after the use of RB, sunlight exposure may avoid to prevent from its harmful effects.

Subretinal fluid is common in experimental non-arteritic anterior ischemic optic neuropathy.

PURPOSE: Anterior ischemic optic neuropathy (AION) is an important cause of acute vision loss for which several animal models exist. It has been associated with subretinal fluid in a previous study on patients but not yet so in animal models. PATIENTS AND METHODS: A patient presented with acute non-arteritic AION (NAION) and underwent ophthalmic evaluation and testing including fluorescein angiography and spectral-domain optical coherence tomography (SD-OCT). On the basis of the patient's findings, we used SD-OCT circular and volume scans to analyze retinal changes in a murine model of NAION. RESULTS: One week after left eye vision loss, the patient had clinical and imaging findings consistent with NAION. On SD-OCT, there was prominent peripapillary retinal thickening consistent with intra-retinal edema and sub-foveolar fluid. Inspired by the findings in human AION, we looked for similar changes in murine NAION using SD-OCT. The circular scan did not adequately detect the presence of subretinal fluid. Using the 25-line scan, which covered a larger part of the posterior pole, we found that 100% of murine AION resulted in subretinal fluid at day 1. The subretinal fluid resolved by week 1. CONCLUSION: This study detailed a case of clinical NAION associated with intra-retinal and subretinal fluid. We also found that subretinal fluid was common in murine photochemical thrombosis model of AION and could be found far away from the optic disc.

Antimicrobial activity and cytotoxicity of 3 photosensitizers activated with blue light.

INTRODUCTION: Pulp repair is less likely to occur when dentin or pulpal tissue remains infected after caries excavation. Yet there are currently few options to kill residual bacteria without damaging resident cells. The current study has evaluated the effect of 3 blue light-activated chemicals on the viability of lactobacilli, odontoblast-like cells (MDPC-23), undifferentiated pulp cells (OD21), and human embryonic stem cells (hESC H1). METHODS: Bacteria were incubated for 15 minutes with curcumin, eosin Y, or rose bengal and then irradiated with blue light (240 seconds). Bacteria were labeled with LIVE/DEAD BacLight Bacterial Viability kit, and viability was assessed by fluorescence-activated cell sorting. Cytotoxicity assays were performed on MDPC-23 cells, OD21, and hESC H1 cells grown in 24-well plates and exposed to the same photosensitizer-light combination. After 24 hours, cellular response was measured by using the methyl-thiazol-diphenyl-tetrazolium assay. Results were statistically analyzed by using one-way analysis of variance and Tukey multiple comparison intervals (α = 0.05). RESULTS: Bacterial viability was significantly reduced after exposure to different combinations of light and photosensitizers; mitochondrial activity of cultured cells remained unaffected when exposed to the same conditions, suggesting a good therapeutic index in vitro. CONCLUSIONS: Blue light-mediated disinfection is promising for the development of new treatment strategies designed to promote pulp repair after carious exposure.

Rutin improves functional outcome via reducing the elevated matrix metalloproteinase-9 level in a photothrombotic focal ischemic model of rats.

BACKGROUND: Blood-brain barrier (BBB) disruption mediated by proteases plays a pivotal role in neural tissue damage after acute ischemic stroke. In an animal stroke model, the activation of matrix metalloproteinases (MMPs), especially MMP-9, was significantly increased and it showed potential association with blood-brain barrier (BBB) disruption and cerebral edema. Theoretically, it is expected that early blockade of expression and activation of MMP-9 after ischemic stroke provides neuroprotective effects from secondary neural tissue damage. This study was aimed to determine the ability of rutin to influence MMP-9 expression, activity and BBB disruption using a photothrombotic focal ischemic model in rats. METHODS: Adult male Sprague-Dawley rats, weighing between 250 and 300 g (aged 8 weeks) received focal cerebral ischemia by photothrombosis using Rose Bengal (RB) and cold light. Injured animals were divided into two groups; one group received 50mg/kg of rutin intraperitoneally, starting 1h after injury and at 12h intervals for 3 days, while animals in the control group received weight-adjusted doses of saline vehicle over the same period. In each group, the expressions and activities of MMP-9 were assessed by Western blot and gelatin zymography at 6, 24, 48, and 72 h after photothrombotic insult. The effects of rutin on BBB disruption and functional outcomes were also determined. RESULTS: Western blot and zymographic analysis showed up-regulated MMP-9 expression and activity in the ischemic cortex. The expression and activity of MMP-9 were significantly elevated at 6h after photothrombotic insult, which remained up-regulated for at least until 72 h after injury. In the rutin-treated group, MMP-9 expression and activity were significantly attenuated at 6, 24, and 48 h compared to the control group. Relative to the control group, BBB permeability was significantly reduced in the rutin-treated group. The results of the rotarod test revealed that rutin treatment significantly improved functional outcomes. CONCLUSIONS: Rutin treatment starting 1h after injury attenuated BBB disruption during photothrombotic focal ischemia, which was partly, at least, achieved through inhibitory effects on MMP-9 expression and activity. The results of this study suggest that rutin might be useful in clinical trials aimed to improve the outcome of patients suffering from acute ischemic stroke.

Functional assessment of sensory functions after photothrombotic stroke in the barrel field of mice.

Motor, sensory and cognitive deficits are common impairments observed in human stroke as well as in animal stroke models. Using a battery of behavioural tests we assessed sensorimotor deficits after photothrombotic stroke localized within or beyond cortical representation of mouse sensory vibrissae. We found restricted, modality specific behavioural consequences in the acute post-stroke period. Among incorporated tests, adhesive removal test, novelty exploration test and sensory labyrinth task were sensitive to the somatosensory cortical deficits. Injured animals explored new objects significantly longer, they also needed distinctly more time to contact and to remove the adhesive tape placed on whiskers contralateral to the infarct. Moreover, we observed that after stroke animals were unable to solve the sensory labyrinth depending only upon tactile sensation from whiskers with injured cortical representation. Spontaneous recovery could be observed within the first post-stroke week for adhesive tape removal and within 14 days for labyrinth performance. However, for the novel object exploration we did not observed the recovery for the period of 18 days after stroke. Moreover, new object exploration test performance differed between the somatosensory and visual cortical impairments. We suggest that those three tests might be valuable in assessing the usefulness of therapies designed to support brain repair after experimental stroke.

Why is rose bengal more phototoxic to fibroblasts in vitro than in vivo?

Photosensitized protein cross-linking has been recently developed to seal wounds and strengthen tissue. Although the photosensitizing dye, Rose Bengal (RB), is phototoxic to cultured cells, cytotoxicity does not accompany RB-photosensitized tissue repair in vivo. We investigated whether the environment surrounding cells in tissue or the high irradiances used for photo-cross-linking inhibited RB phototoxicity. Fibroblasts (FB) grown within collagen gels to mimic a tissue environment and monolayer cultured FB were treated with RB (0.01-1 mm) and the high 532 nm laser irradiances used in vivo for tissue repair (0.10-0.50 W cm(-2)). Monolayer FB were substantially more sensitive to RB photosensitization: the LD50 was >200-fold lower than that in collagen gels. Collagen gel protection was associated with increased Akt phosphorylation, a prosurvival pathway. RB phototoxicity in collagen gels was 25-fold greater at low (0.030 W cm(-2)) that at high (0.50 W cm(-2)) irradiances. Oxygen depletion at high irradiance only partially accounted for the irradiance dependence of phototoxicity as replacing air with nitrogen only increased the LD50 by four-fold in monolayers. These results indicate that the lack of RB phototoxicity during in vivo tissue repair results from upregulation of prosurvival pathways in tissue cells, oxygen depletion and irradiance-dependent RB photochemistry.

Electrophysiological correlations of morphological restructuring in experimental local ischemia of different severity in the rat sensorimotor cortex.

Local cerebrovascular disorders were modeled by reversible photochemical clotting of hemispheric cortical vessels. Mild ischemia led to reversible edema in the surface layers of the cortex: cytotoxic edema of the neuropile, primarily of the distal dendrites. This status led to an increase in the lower delta rhythm frequency band power. After administration of systemic anesthetic, delta rhythm appeared sooner in the ischemic foci than in intact cortical areas. More severe ischemia led to the appearance of dark and pyknotic neurons and reduction of oscillation power in all EEG spectrum bands. Restructuring of primarily dendrites caused by local moderate ischemia of the surface cortical layers at the early stage of neurodegenerative processes stimulated the inhibitory recovery processes.

A new non-human primate model of photochemically induced cerebral infarction.

BACKGROUND AND PURPOSE: Rat models of photochemically induced cerebral infarction have been readily studied, but to date there are no reports of transcranial photochemically induced infarctions in the marmoset. In this report, we used this non-human primate as a model of cerebral thrombosis and observed the recovery process. METHODS: Five common marmosets were used. Cerebral ischemia was produced via intravascular thrombosis induced by an intravenous injection of Rose Bengal and irradiation with green light. After inducing cerebral infarction, we observed the behavior of marmosets via a continuous video recording. We evaluated maximum speed, mean speed, and distance traveled in 1 min. In addition, we evaluated scores for feeding behavior, upper limb grip, and lower limb grip. We confirmed the infarct area after cerebral infarction using 2,3,5-triphenyltetrazolium chloride staining in a separate marmoset. RESULTS: We found functional decreases 2 days after creating the cerebral infarction in all measurements. Total distance traveled, average speed, upper limb score, and feeding behavior score did not recover to pre-infarction levels within 28 days. Maximum speed in 1 min and lower limb score recovered 28 days after infarction as compared to pre-infarction levels. We confirmed the infarct area of 11.4 mm × 6.8 mm as stained with 2,3,5-triphenyltetrazolium chloride. CONCLUSION: We were able to create a primate photothrombosis-induced cerebral infarction model using marmosets and observe functional recovery. We suggest that this is a useful model for basic research of cerebral infarction.

Comparative analysis of four oxidized guanine lesions from reactions of DNA with peroxynitrite, singlet oxygen, and γ-radiation.

Oxidative damage to DNA has many origins, including irradiation, inflammation, and oxidative stress, but the chemistries are not the same. The most oxidizable base in DNA is 2-deoxyguanosine (dG), and the primary oxidation products are 8-oxodG and 2-amino-imidazolone. The latter rapidly converts to 2,2-diamino-oxazolone (Ox), and 8-oxodG is further oxidized to spiroiminodihydantoin (Sp) and guanidinohydantoin (Gh). In this study, we have examined the dose-response relationship for the formation of the above four products arising in calf thymus DNA exposed to gamma irradiation, photoactivated rose bengal, and two sources of peroxynitrite. In order to carry out these experiments, we developed a chromatographic system and synthesized isotopomeric internal standards to enable accurate and precise analysis based upon selected reaction monitoring mass spectrometry. 8-OxodG was the most abundant products in all cases, but its accumulation was highly dependent on the nature of the oxidizing agent and the subsequent conversion to Sp and Gh. Among the other oxidation products, Ox was the most abundant, and Sp was formed in significantly greater yield than Gh.

Melatonin reduced the elevated matrix metalloproteinase-9 level in a rat photothrombotic stroke model.

BACKGROUND: The accumulation of toxic free radicals plays a pivotal role in the early molecular cascades of blood-brain barrier (BBB) disruption mediated by matrix metalloproteinases (MMPs) activation in ischemic stroke. Theoretically, it is expected that early blockade of activation of MMPs may provide protective effects from secondary neural tissue damage. The present study was designed to determine the ability of melatonin to influence MMP-9 activity and BBB disruption, in a focal ischemia rat model induced by photothrombosis. METHODS: Adult, male, 8-week Sprague-Dawley rats weighing 230-300 g received focal cerebral ischemia by photothrombosis using Rose Bengal (RB). The injured animals were divided into two groups. One group received 50mg/kg of melatonin intraperitoneally, starting 1h after injury and at 12h intervals for 3 days. The control group received weight-adjusted doses of saline vehicle. In each group, MMP-9 expression and activity were assessed by Western blot and gelatin zymography, respectively, at various times. The effects of melatonin on BBB disruption and brain edema were also determined. RESULTS: MMP-9 activity and expression were significantly elevated at 24h in the ischemic cortex, which remained up-regulated at least until 72 h after injury. Melatonin treatment significantly attenuated MMP-9 activity and expression at 24, 48, and 72 h after ischemic injury. Relative to control group, BBB permeability was significantly reduced in the melatonin-treated group. The water content was decreased by melatonin treatment, although there was no statistically significant difference. CONCLUSIONS: Melatonin treatment starting 1h after injury attenuated BBB disruption during focal ischemia, which is at least partly due to inhibition of MMP-9 activity. Melatonin might have a potential role in clinical trials aimed to improve the outcome of patients suffering cerebral ischemia.

Cellular inflammation in nonarteritic anterior ischemic optic neuropathy and its primate model.

OBJECTIVE: To correlate potential inflammatory responses in nonarteritic anterior ischemic optic neuropathy (NAION) with a lesion possessing many physiologic and histologic similarities from a model of nonhuman primate NAION (pNAION). METHODS: Using immunohistochemistry and confocal microscopic analysis, we evaluated the relative numbers of inflammatory cell types in the single available clinical specimen of early NAION (21 days after event). We correlated this with the temporal inflammatory response occurring in optic nerve tissue at different times following pNAION induction. RESULTS: In pNAION, there is a previously unsuspected infiltration of polymorphonuclear leukocytes occurring almost immediately after infarct induction, followed by invasion of ED1+ extrinsic macrophages, which peaks 5 weeks after infarct. Intrinsic microglia accumulate up to 70 days after induction in the area of primary axonal loss. The analyzed human NAION specimen was similar to 21-day pNAION tissue, with extrinsic macrophages and intrinsic microglial cells in the region of focal axon loss. CONCLUSIONS: Cellular inflammation plays a major early role following white-matter (optic nerve) infarct, with both polymorphonuclear leukocyte and macrophage function involved in debris elimination and tissue remodeling. The optic nerve in NAION and its primate model are associated with early cellular inflammation, previously unsuspected, that may contribute to postinfarct optic nerve damage.

Neuronal gap junctions are required for NMDA receptor-mediated excitotoxicity: implications in ischemic stroke.

N-methyl-D-aspartate receptors (NMDARs) play an important role in cell survival versus cell death decisions during neuronal development, ischemia, trauma, and epilepsy. Coupling of neurons by electrical synapses (gap junctions) is high or increases in neuronal networks during all these conditions. In the developing CNS, neuronal gap junctions are critical for two different types of NMDAR-dependent cell death. However, whether neuronal gap junctions play a role in NMDAR-dependent neuronal death in the mature CNS was not known. Using Fluoro-Jade B staining, we show that a single intraperitoneal administration of NMDA (100 mg/kg) to adult wild-type mice induces neurodegeneration in three forebrain regions, including rostral dentate gyrus. However, the NMDAR-mediated neuronal death is prevented by pharmacological blockade of neuronal gap junctions (with mefloquine, 30 mg/kg) and does not occur in mice lacking neuronal gap junction protein, connexin 36. Using Western blots, electrophysiology, calcium imaging, and gas chromatography-mass spectrometry in wild-type and connexin 36 knockout mice, we show that the reduced level of neuronal death in knockout animals is not caused by the reduced expression of NMDARs, activity of NMDARs, or permeability of the blood-brain barrier to NMDA. In wild-type animals, this neuronal death is not caused by upregulation of connexin 36 by NMDA. Finally, pharmacological and genetic inactivation of neuronal gap junctions in mice also dramatically reduces neuronal death caused by photothrombotic focal cerebral ischemia. The results indicate that neuronal gap junctions are required for NMDAR-dependent excitotoxicity and play a critical role in ischemic neuronal death.

Axonal degeneration, regeneration and ganglion cell death in a rodent model of anterior ischemic optic neuropathy (rAION).

Using laser-induced photoactivation of intravenously administered rose Bengal in rats, we generated an ischemic infarction of the intrascleral portion of the optic nerve (ON) comparable to that which occurs in humans to investigate optic nerve axon degenerative events following optic nerve infarct and the potential for axon re-growth. Animals were euthanized at different times post infarct. Axon degeneration was evaluated with SMI312 immunolabeling, and GAP-43 immunostaining was used to identify axon regeneration. Terminal dUTP nick end labeling (TUNEL) was used to evaluate retinal ganglion cell (RGC) death. There was significant axon structural disruptinot ion at the anterior intrascleral portion of the ON by 3d post-infarct, extending to the posterior ON by 7d post-stroke. Destruction of normal axon structure and massive loss of axon fibers occurred by 2 weeks. GAP-43 immunoreactivity occurred in the anterior ON by 7d post-infarct, lasting 3-4 weeks, without extension past the primary ischemic lesion. TUNEL-positive cells in the RGC layer appeared by 7d post-insult. These results indicate that following induction of ischemic optic neuropathy, significant axon damage occurs by 3d post-infarct, with later neuronal death. Post-stroke adult rat retinal ganglion cells attempt to regenerate their axons, but this effort is restricted to the unmyelinated region of the anterior ON. These responses are important in understanding pathologic process that underlies human non-arteritic anterior ischemic optic neuropathy (NAION) and may guide both the appropriate treatment of NAION and the window of opportunity for such treatment.