Combined treatment of human mesenchymal stem cells and green tea extract on retinal ganglion cell regeneration in rats after optic nerve injury.

Retinal ganglion cell (RGC) death and axonal loss cause irreversible vision loss upon optic nerve (ON) injury. We have independently demonstrated that mesenchymal stem cells (MSCs) and green tea extract (GTE) promote RGC survival and axonal regeneration in rats with ON injury. Here we aimed to evaluate the combined treatment effect of human bone marrow-derived MSCs (hBM-MSCs) and GTE on RGC survival and axonal regeneration after ON injury. Combined treatment of hBM-MSCs and GTE promoted RGC survival and neurite outgrowth/axonal regeneration in ex vivo retinal explant culture and in rats after ON injury. GTE increased Stat3 activation in the retina after combined treatment, and enhanced brain-derived neurotrophic factor secretion from hBM-MSCs. Treatment of 10 µg/mL GTE would not induce hBM-MSC apoptosis, but inhibited their proliferation, migration, and adipogenic and osteogenic differentiation in vitro with reducing matrix metalloproteinase secretions. In summary, this study revealed that GTE can enhance RGC protective effect of hBM-MSCs, suggesting that stem cell priming could be a prospective strategy enhancing the properties of stem cells for ON injury treatment.

Acute direct traumatic optic neuropathy treated with steroids, minocycline and hyperbaric oxygen: a case report.

We describe the emergency management of a man who experienced acute vision loss diagnosed as direct traumatic optic neuropathy (TON) in his right eye (no light perception) after falling from a height. TON is caused by a high-impact mechanism of injury. Clinical findings include acute vision loss, which is typically immediate, afferent pupillary defect, decreased color vision, and visual field defects. Treatment is controversial because of the lack of strong evidence supporting intervention over observation. In this case report, our treatment strategy comprised immediate hyperbaric oxygen (HBO2) and daily high doses of a steroid. On the second day, minocycline was added to the treatment regimen for its neuroprotective effects. The patient was discharged after receiving six HBO2 treatments and six days of intravenous solumedrol transitioned to oral prednisone. After the third HBO2 treatment, his vision improved to 20/100; after the fourth treatment, it was 20/40 and plateaued. At the time of discharge, it was 20/40. At two-month follow-up, his corrected visual acuity was 20/60+2 in the affected eye. Immediate HBO2 for ischemic and mechanical injury to the optic nerve following trauma is a therapeutic option.

Electrical brain stimulation induces dendritic stripping but improves survival of silent neurons after optic nerve damage.

Repetitive transorbital alternating current stimulation (rtACS) improves vision in patients with chronic visual impairments and an acute treatment increased survival of retinal neurons after optic nerve crush (ONC) in rodent models of visual system injury. However, despite this protection no functional recovery could be detected in rats, which was interpreted as evidence of "silent survivor" cells. We now analysed the mechanisms underlying this "silent survival" effect. Using in vivo microscopy of the retina we investigated the survival and morphology of fluorescent neurons before and after ONC in animals receiving rtACS or sham treatment. One week after the crush, more neurons survived in the rtACS-treated group compared to sham-treated controls. In vivo imaging further revealed that in the initial post-ONC period, rtACS induced dendritic pruning in surviving neurons. In contrast, dendrites in untreated retinae degenerated slowly after the axonal trauma and neurons died. The complete loss of visual evoked potentials supports the hypothesis that cell signalling is abolished in the surviving neurons. Despite this evidence of "silencing", intracellular free calcium imaging showed that the cells were still viable. We propose that early after trauma, complete dendritic stripping following rtACS protects neurons from excitotoxic cell death by silencing them.

Alternating Current Stimulation for Vision Restoration after Optic Nerve Damage: A Randomized Clinical Trial.

BACKGROUND: Vision loss after optic neuropathy is considered irreversible. Here, repetitive transorbital alternating current stimulation (rtACS) was applied in partially blind patients with the goal of activating their residual vision. METHODS: We conducted a multicenter, prospective, randomized, double-blind, sham-controlled trial in an ambulatory setting with daily application of rtACS (n = 45) or sham-stimulation (n = 37) for 50 min for a duration of 10 week days. A volunteer sample of patients with optic nerve damage (mean age 59.1 yrs) was recruited. The primary outcome measure for efficacy was super-threshold visual fields with 48 hrs after the last treatment day and at 2-months follow-up. Secondary outcome measures were near-threshold visual fields, reaction time, visual acuity, and resting-state EEGs to assess changes in brain physiology. RESULTS: The rtACS-treated group had a mean improvement in visual field of 24.0% which was significantly greater than after sham-stimulation (2.5%). This improvement persisted for at least 2 months in terms of both within- and between-group comparisons. Secondary analyses revealed improvements of near-threshold visual fields in the central 5° and increased thresholds in static perimetry after rtACS and improved reaction times, but visual acuity did not change compared to shams. Visual field improvement induced by rtACS was associated with EEG power-spectra and coherence alterations in visual cortical networks which are interpreted as signs of neuromodulation. Current flow simulation indicates current in the frontal cortex, eye, and optic nerve and in the subcortical but not in the cortical regions. CONCLUSION: rtACS treatment is a safe and effective means to partially restore vision after optic nerve damage probably by modulating brain plasticity. This class 1 evidence suggests that visual fields can be improved in a clinically meaningful way. TRIAL REGISTRATION: ClinicalTrials.gov NCT01280877.

Repetitive Transcorneal Alternating Current Stimulation Reduces Brain Idling State After Long-term Vision Loss.

BACKGROUND: Deafferentation of visual system structures following brain or optic nerve injury leaves cortical areas deprived of visual input. Deprived cortical areas have a reduced sensory information processing and are characterized with localized enhanced or synchronized rhythms believed to represent an "idling state". OBJECTIVE/HYPOTHESIS: We hypothesized that cortical idling can be modified with transcorneal alternating current stimulation (tACS) known to modulate cortical oscillations and thus change the functional state of the deafferented areas. METHODS: tACS was applied in rat model of severe optic nerve crush using a protocol similar to our clinical studies (200 µA, 2-8 Hz) for 5 treatment days right after the lesion and at the chronic stage (3 months later). EEG and VEP were recorded over the visual cortices. In vivo confocal neuroimaging of the retina and histology of the optic nerves were performed. RESULTS: Morphological investigations showed massive retinal ganglion cells death and degeneration of the optic nerves after crush. Visual loss was associated with increased EEG spectral power and lower coherence, indicating an "idling state". Stimulation induced a significant decrease of EEG power towards normal values. These effects were especially pronounced in the chronic stage. CONCLUSION: Our results suggest that alternating current injected via the eye is able to modulate visually deprived brain areas and thus reduce cortical idling.

Effect of Lycium barbarum (Wolfberry) on alleviating axonal degeneration after partial optic nerve transection.

Our previous results showed that the polysaccharides extracted from Lycium barbarum (LBP) could delay secondary degeneration of retinal ganglion cell bodies and improve the function of the retinas after partial optic nerve transection (PONT). Although the common degeneration mechanisms were believed to be shared by both neuronal bodies and axons, recently published data from slow Wallerian degeneration mutant (Wld(s)) mice supported the divergence in the mechanisms of them. Therefore, we want to determine if LBP could also delay the degeneration of axons after PONT. Microglia/macrophages were thought to be a source of reactive oxygen species after central nervous system (CNS) injury. After PONT, however, oxidative stress was believed to occur prior to the activation of microglia/macrophages in the areas vulnerable to secondary degeneration both in the optic nerves (ONs) and the retinas. But the results did not take into account the morphological changes of microglia/macrophages after their activation. So we examined the morphology in addition to the response magnitude of microglia/macrophages to determine their time point of activation. In addition, the effects of LBP on the activation of microglia/macrophages were investigated. The results showed that (1) LBP reduced the loss of axons in the central ONs and preserved the g-ratio (axon diameter/fiber diameter) in the ventral ONs although no significant effect was detected in the dorsal ONs; (2) microglia/macrophages were activated in the ONs by 12 h after PONT; (3) LBP decreased the response magnitude of microglia/macrophages 4 weeks after PONT. In conclusion, our results showed that LBP could delay secondary degeneration of the axons, and LBP could also inhibit the activation of microglia/macrophages. Therefore, LBP could be a promising herbal medicine to delay secondary degeneration in the CNS via modulating the function of microglia/macrophages.

Paranode Abnormalities and Oxidative Stress in Optic Nerve Vulnerable to Secondary Degeneration: Modulation by 670 nm Light Treatment.

Secondary degeneration of nerve tissue adjacent to a traumatic injury results in further loss of neurons, glia and function, via mechanisms that may involve oxidative stress. However, changes in indicators of oxidative stress have not yet been demonstrated in oligodendrocytes vulnerable to secondary degeneration in vivo. We show increases in the oxidative stress indicator carboxymethyl lysine at days 1 and 3 after injury in oligodendrocytes vulnerable to secondary degeneration. Dihydroethidium staining for superoxide is reduced, indicating endogenous control of this particular reactive species after injury. Concurrently, node of Ranvier/paranode complexes are altered, with significant lengthening of the paranodal gap and paranode as well as paranode disorganisation. Therapeutic administration of 670 nm light is thought to improve oxidative metabolism via mechanisms that may include increased activity of cytochrome c oxidase. Here, we show that light at 670 nm, delivered for 30 minutes per day, results in in vivo increases in cytochrome c oxidase activity co-localised with oligodendrocytes. Short term (1 day) 670 nm light treatment is associated with reductions in reactive species at the injury site. In optic nerve vulnerable to secondary degeneration superoxide in oligodendrocytes is reduced relative to handling controls, and is associated with reduced paranode abnormalities. Long term (3 month) administration of 670 nm light preserves retinal ganglion cells vulnerable to secondary degeneration and maintains visual function, as assessed by the optokinetic nystagmus visual reflex. Light at a wavelength of 670 nm may serve as a therapeutic intervention for treatment of secondary degeneration following neurotrauma.

Repetitive transorbital alternating current stimulation in optic neuropathy.

BACKGROUND: Visual field defects after optic nerve damage typically show a limited capacity for spontaneous and treatment-induced recovery. OBJECTIVE: Repetitive transorbital alternating current stimulation (rtACS) was applied to the damaged optic nerve to evaluate visual functions after stimulation. METHODS: A 27-years-old male patient suffering left optic nerve atrophy with nearly complete loss of vision 11 years after atypical traumatic damage was treated transorbitally with biphasic 10-15 pulse trains of rtACS (10-30 Hz, < 600 µA, 30-40 min daily for 10 days) which produced phosphenes. RESULTS: After rtACS treatment detection ability of super-threshold stimuli increased from 3.44% to 17.75% and mean perimetric threshold from 0 dB to 2.21 dB at final diagnostics. CONCLUSION: This improvement of vision may be due to increased neuronal synchronization, possibly involving strengthening of synaptic transmission along the central visual pathway.

Neuroprotective effect of transpupillary thermotherapy in the optic nerve crush model of the rat.

PURPOSE: Transpupillary thermotherapy (TTT) has been shown to induce heat shock protein (Hsp) 72 in optic nerve head tissue. The neuroprotective effect of TTT was investigated in an optic nerve crush rat model. METHODS: TTT was performed onto the optic nerve head in the right eye of subject rats. After 24 h, an optic nerve crush injury using an aneurysm clip was performed at 2 mm from the optic nerve head for 60 s. At 7 and 14 days later, retrograde labelling of retinal ganglion cells (RGCs) with DTMR crystal was carried out and the density of the surviving RGCs was evaluated. Immunohistochemical staining was performed to confirm the expression of Hsp72. RESULTS: At 7 days after optic nerve crush injury, the mean density of surviving RGCs was higher in TTT group (372.7+/-149.8 per mm(2)) than in optic nerve crush group (252.9+/-96.7 per mm(2)) with borderline significance. In the retinal areas at 1 mm from the optic nerve head, a significant increase in surviving RGCs from TTT treated eyes was observed at both 7 and 14 days after optic nerve crush injury. However, no significant differences in surviving RGCs were demonstrated 2 and 3 mm from the optic nerve head. CONCLUSIONS: These results demonstrate that TTT aimed onto the optic nerve head showed a neuroprotective effect.

Bone marrow-derived mesenchymal stem cell transplantation does not improve quality of muscle reinnervation or recovery of motor function after facial nerve transection in rats.

Recently, we devised and validated a novel strategy in rats to improve the outcome of facial nerve reconstruction by daily manual stimulation of the target muscles. The treatment resulted in full recovery of facial movements (whisking), which was achieved by reducing the proportion of pathologically polyinnervated motor endplates. Here, we posed whether manual stimulation could also be beneficial after a surgical procedure potentially useful for treatment of large peripheral nerve defects, i.e., entubulation of the transected facial nerve in a conduit filled with suspension of isogeneic bone marrow-derived mesenchymal stem cells (BM-MSCs) in collagen. Compared to control treatment with collagen only, entubulation with BM-MSCs failed to decrease the extent of collateral axonal branching at the lesion site and did not improve functional recovery. Post-operative manual stimulation of vibrissal muscles also failed to promote a better recovery following entubulation with BM-MSCs. We suggest that BM-MSCs promote excessive trophic support for regenerating axons which, in turn, results in excessive collateral branching at the lesion site and extensive polyinnervation of the motor endplates. Furthermore, such deleterious effects cannot be overridden by manual stimulation. We conclude that entubulation with BM-MSCs is not beneficial for facial nerve repair.

Parameters of optic nerve electrical stimulation affecting neuroprotection of axotomized retinal ganglion cells in adult rats.

We previously showed the enhancement of survival of retinal ganglion cells (RGCs) by electrical stimulation (ES) of the optic nerve (ON) stump in adult rats. To elucidate the mechanisms underlying the survival enhancement, we determined whether the neuroprotective effect of ES is affected by the following parameters: stimulation time, frequency of current pulses and starting of ES. ES for 10min immediately after ON transection was not effective in increasing the number of surviving RGCs 7 days after the transection, but that for 30min was effective. ES at 20Hz was the most effective, when applied just after axotomy. When the starting of ES to the ON was shifted either 3h after or 4h before the axotomy, the neuroprotective effect of ES was not observed. These results suggest that the electrical activation of RGCs and/or the transected ON interfere with early events after axotomy that leads to RGC death.

Neuroprotective effect of transcorneal electrical stimulation on the acute phase of optic nerve injury.

PURPOSE: Traumatic optic neuropathy often induces a loss of vision that proceeds rapidly within several hours, together with retinal ganglion cell death, in a much slower time course. Electrical stimulation has previously been shown to rescue injured retinal ganglion cells from cell death. The present study tests whether transcorneal electrical stimulation could preserve visual function after an optic nerve crush. METHODS: Transcorneal electrical stimulation was given immediately after a calibrated optic nerve crush. We measured visually evoked potentials (VEPs) in the visual cortex of rats before and immediately after the optic nerve crush and after the transcorneal stimulation to estimate an extent of damage and effects of stimulation in individual animals. In addition, the retinal axons were labeled with a fluorescent anterograde tracer to determine whether the transcorneal electrical stimulation can protect the retinal axons from degeneration. RESULTS: The optic nerve crush was made at an intensity that does not allow a spontaneous recovery of VEP for 1 week. The transcorneal stimulation immediately increased VEP amplitude impaired by the optic nerve crush, and this augmentation was often preserved after 1 week. In the stimulated animals, a larger amount of retinal axons projected centrally beyond the crushed region in comparison to the unstimulated animals. CONCLUSIONS: Transcorneal electrical stimulation would restore the functional impairment of optic nerve by traumatic injury at a very early stage and protect retinal axons from the ensuing degeneration.

Effect of transcorneal electrical stimulation in patients with nonarteritic ischemic optic neuropathy or traumatic optic neuropathy.

PURPOSE: To determine whether transcorneal electrical stimulation (TES) can improve the visual function of patients with nonarteritic ischemic optic neuropathy (NAION) or traumatic optic neuropathy (TON). METHODS: Eight consecutive patients at the Osaka University Hospital were studied. TES (600-800 microA, 20 Hz, 30 min) was applied once each to three eyes with NAION and to five eyes with TON, using a contact lens-type stimulating electrode. The primary outcome measurement was the change in visual acuity at 1 to 3 months after TES. An improvement in visual acuity was defined as a change of > or =0.3 log (minimum angle of resolution) (logMAR) units. The side effects of TES were also investigated. RESULTS: After TES application, the visual acuity improved in two patients with NAION and in four patients with TON. Visual acuity did not worsen in any of the eyes. Only a mild superficial punctuate keratopathy was observed in all eyes immediately after TES, and it healed by the next day. CONCLUSIONS: Visual acuity can be improved after TES without major complications in some patients with NAION or TON. These results suggest that TES should be considered as a new treatment for eyes with optic neuropathy.