Zebrafish (Danio rerio) Developed as an Alternative Animal Model for Focal Ischemic Stroke.

Thrombotic cerebral ischemia is one of the leading causes of mortality and chronic disability. Animal models provide an essential tool for understanding the complex cellular and molecular pathophysiology of ischemia and for improving treatment and testing novel neuroprotective drugs in the preclinical setting. In this study, we tested zebrafish as a novel model for thrombotic ischemic brain damage. Zebrafish were intraperitoneally injected with Rose Bengal and light exposure was directed onto the optic tectum region of the brain to induce photothrombosis. After full recovery from anesthesia, zebrafish consistently exhibited abnormal swimming patterns, indicating brain injury from the procedure. The staining of 2,3,5-triphenyltetrazolium chloride (TTC) 24 h after the treatment showed lack of staining of the exposed area of the brain, which further confirmed the ischemic injury. Application of Activase®-tPA improved viability of the brain. The tPA treatment also reduced the occurrence of moving disability as well as the mortality rate, demonstrating that the zebrafish model not only showed focal ischemic injury but also responded well to tPA therapy. Our results suggest that the current photothrombotic method induced focal ischemia in zebrafish and produced consistent brain damage that can be measured by behavioral changes and quantified by histological staining.

Analysis of Small Ischemic Lesions in the Examinees of a Brain Dock and Neurological Examination of Animals Subjected to Cortical or Basal Ganglia Photothrombotic Infarction.

We analyzed cases of small brain ischemic lesions found in examinees of a brain dock (neurological health screening center). Small cerebral infarction was found in 17 % of the examinees (733 cases). White matter lesions were found in 24 %. Infarctions were located in the cortex or subcortical white matter in 31 % and in the basal ganglia in 44 % of cases. Infratentorial infarction was found in 1.6 %. We have developed an animal model of small infarction in the cortex or basal ganglia induced by photothrombosis in rodents. Sprague-Dawley rats or Mongolian gerbils were anesthetized and photothrombotic infarction was induced in the left caudate nucleus or parietal cortex by light exposure via an optic fiber and intravenous Rose Bengal dye injection. Histological examination revealed development of a small spherical infarction surrounding the tip of the optic fiber. The lesion turned to a cyst by 6 weeks after lesioning. Neurological deficits were found in animals both with cortical and caudate infarction. Behavioral changes in an open field test differed with the lesion site. Neurological deficits were sustained longer in animals with larger infarctions. Thus, photothrombotic infarction is useful for analyzing location-dependent and size-dependent neurological and neuropathological changes after cerebral infarction.

Topical liposomal Rose Bengal for photodynamic white hair removal: randomized, controlled, double-blind study.

BACKGROUND: Blond and white hair removal by laser is a complicated task with weak satisfactory results due to the deficiency in laser-absorbing chromophore. OBJECTIVE: To investigate if repetitive sessions of photodynamic therapy (PDT) using external application of liposomal Rose bengal (RB) photosensitizer followed by intense pulsed light (IPL) exposure enables removal of gray and white hair. MATERIALS AND METHODS: Rose bengal loaded in liposomes (LRB) was constructed, prepared in hydrogel, and was studied for some pharmaceutical properties. Penetration and selective hair follicle damage in mice skin were studied. Topical gel containing LRB was used for treating fifteen adult females who were complaining of facial white terminal hair. Unwanted facial hair was treated for three sessions at intervals of 4-6 weeks using intense pulsed light (IPL). At each session, the treatment area was pre-treated with topical LRB gel, while a control group of another 15 patients applied placebo gel before IPL treatment. Evaluations included hair regrowth, which was measured 4 weeks after each treatment session and at 6 months follow-up by counting the number of terminal hair compared with baseline pretreatment values. Treatment outcomes and complications if any were also reported. RESULTS: Average hair regrowth in the LRB group was 56% after 3 treatment cycles. After six-months follow up, average terminal hair count compared with baseline pretreatment showed 40% reduction and no recorded side effects. A significant difference (P<0.05) was seen compared with the control group; the clinical results were promising. CONCLUSIONS: Photodynamic hair removal using rose bengal-encapsulated liposomal gel in combination with IPL treatment showed significant efficacy in the treatment of white hair compared with a control group.

In vitro cell compatibility study of rose bengal-chitosan adhesives.

BACKGROUND AND OBJECTIVES: Photochemical tissue bonding (PTB) using rose bengal (RB) in conjunction with light is an alternative technique to repair tissue without suturing. It was recently demonstrated that laser-irradiated chitosan films, incorporating RB, bonded firmly to calf intestine in vitro. It is thus required to investigate the possible cytotoxic effects of the RB-chitosan adhesive on cells before testing its application to in vivo models. MATERIALS AND METHODS: Adhesive films, based on chitosan and containing ~0.1 wt% RB were fabricated. Their cytotoxicity was assessed by growing human and murine fibroblasts either in media in which adhesive strips had been incubated, or directly on the adhesive. The adhesive was either laser-irradiated or not. Cells were stained after 48 hours with Trypan blue and the number of live and dead cells was recorded for cell viability. RESULTS: Murine and human fibroblasts grew confluent on the adhesives with no apparent morphological changes or any exclusion zone. Cell numbers of murine fibroblasts were not significantly different when cultured in media that was extracted from irradiated (86 ± 7%) and non-irradiated adhesive (89 ± 4%). A similar result was obtained for the human fibroblasts. CONCLUSIONS: These findings support that the RB-chitosan films induced negligible toxicity and growth retardation in murine and human fibroblasts.

A Quantitative Model for Estimating the Scale of Photochemically Induced Ischemic Stroke.

Photothrombosis is a technique that can induce ischemic cortical infarcts using the photodynamic effect of anionic xanthene dyes, typically Rose Bengal, to cause occlusion of cerebral blood circulation. The ability to quantitatively predict the scale of the lesion in photothrombotic procedures can offer crucial insight in the development and implementation of light-induced stroke models in animals. In this article, we introduced a quantitative model that could estimate the normalized light intensity distribution in tissue which scatters photons from a collimated beam. We simulated the penetration and scattering profile of light of Rose Bengal's characteristic absorption wavelengths in mouse cortex. We further illustrated that our model could estimate the spatial extent of effective region under photothrombotic protocols, and how this model can be used to titrate the intensity and geometry of light beams used to generate infarcts of desired dimensional characteristics.

Dynasore protects the ocular surface against damaging oxidative stress.

Water soluble "vital" dyes are commonly used clinically to evaluate health of the ocular surface; however, staining mechanisms remain poorly understood. Recent evidence suggests that sublethal damage stimulates vital dye uptake by individual living cells. Since cell damage can also stimulate reparative plasma membrane remodeling, we hypothesized that dye uptake occurs via endocytic vesicles. In support of this idea, we show here that application of oxidative stress to relatively undifferentiated monolayer cultures of human corneal epithelial cells stimulates both dye uptake and endocytosis, and that dye uptake is blocked by co-treatment with three different endocytosis inhibitors. Stress application to stratified and differentiated corneal epithelial cell cultures, which are a better model of the ocular surface, also stimulated dye uptake; however, endocytosis was not stimulated, and two of the endocytosis inhibitors did not block dye uptake. The exception was Dynasore and its more potent analogue Dyngo-4a, both small molecules developed to target dynamin family GTPases, but also having off-target effects on the plasma membrane. Significantly, while Dynasore blocked stress-stimulated dye uptake at the ocular surface of ex vivo mouse eyes when treatment was performed at the same time as eyes were stressed, it had no effect when used after stress was applied and the ocular surface was already damaged. Thus, Dynasore could not be working by inhibiting endocytosis. Employing cytotoxicity and western blotting assays, we went on to demonstrate an alternative mechanism. We show that Dynasore is remarkably protective of cells and their surface glycocalyx, preventing damage due to stress, and thus precluding dye entry. These unexpected and novel findings provide greater insight into the mechanisms of vital dye uptake and point the direction for future study. Significantly, they also suggest that Dynasore and its analogues might be used therapeutically to protect the ocular surface and to treat ocular surface disease.

Epileptogenesis after cortical photothrombotic brain lesion in rats.

We investigated epileptogenesis after cortical photothrombotic stroke induced with Rose Bengal dye in adult Sprague-Dawley rats. To detect spontaneous seizures, video-electroencephalograms were recorded at 2, 4, 6, 8, and 10 months for 7-14 days (24 h/day). At the end, spatial and emotional learning and memory were assessed using the Morris water-maze and fear-conditioning test, respectively, and the brains were processed for histologic analysis. Seizures were detected in 18% of rats that received photothrombosis. The average seizure frequency was 0.39 seizures per recording day and mean seizure duration was 117 s. Over 60% of seizures occurred during the dark hours. Rats with photothrombotic lesions were impaired in the water-maze (P<0.05) but not in the fear-conditioning test as compared with controls. Histology revealed that lesion depth varied from cortical layers I to VI in photothrombotic rats with epilepsy. Epileptic rats had light mossy fiber sprouting in the inner molecular layer of the dentate gyrus both ipsilateral and contralateral to the lesion. This study extends the current understanding of epileptogenesis and functional impairment after cortical lesions induced by photothrombosis. Our observations support the hypothesis that photothrombotic stroke in rats is a useful animal model for investigating the mechanisms of post-stroke epileptogenesis.

Photochemically induced cerebral ischemia in a mouse model.

BACKGROUND: MCAO has been widely used to produce ischemic brain lesions. The lesions induced by MCAO tend to be variable in size because of the variance in the collateral blood supply found in the mouse brain. METHODS: We modified the rat photothrombosis model for use in mice. Male C57BL/6 mice were subjected to focal cerebral ischemia by photothrombosis of cortical microvessels. Cerebral infarction was produced by intraperitoneal injection of rose bengal, a photosensitive dye, and by focal illumination through the skull. Motor impairment was assessed by the accelerating rotarod and staircase tests. The brain was perfusion fixed for histologic determination of infarct volume 4 weeks after stroke. RESULTS: The lesion was located in the frontal and parietal cortex and the underlying white matter was partly affected. A relatively constant infarct volume was achieved 1 month after photothrombosis. The presence of the photothrombotic lesion significantly impaired the motor performance as measured by the rotarod and staircase tests. Our findings show that photothrombotic infarction in mice is highly reproducible in size and location. CONCLUSION: This procedure can provide a simple model of cerebral infarction for a unilateral motor cortex lesion. In addition, it can provide a suitable model for the study of potential neuroprotective and therapeutic agents in human stroke.

Alterations in anxiety and social behaviour in Npas4 deficient mice following photochemically-induced focal cortical stroke.

In addition to causing widespread cell death and loss of brain function, cerebral ischaemia also induces extensive neuroplasticity. In humans, stroke is often accompanied by severe cognitive and psychiatric changes that are thought to arise as a consequence of this infarct-induced remodelling. A candidate for producing these post-stroke neuropsychiatric changes is Npas4, an activity-dependent transcription factor involved in synaptic plasticity whose expression is aberrantly up-regulated following ischaemic injury. In this study we investigated the role of Npas4 in modulating these stroke-induced neuropsychiatric responses by comparing the performance of wildtype and Npas4-/- mice in various cognitive and behavioural tasks in a photochemical model of focal cortical stroke. We show that this stroke model results in impaired spatial recognition memory and a reduction in despair-like behaviour that affect both genotypes to a similar degree. Moreover, mice lacking Npas4 also show differences in some aspects of post-stroke sociability and anxiety. Specifically, we show that while stroke had no effect on anxiety levels in wildtype mice, Npas4-/- mice became significantly more anxious following stroke. In addition, Npas4-/- mice retained a greater level of sociability in the acute post-stroke period in comparison to their wildtype littermates. Thus, our findings suggest that Npas4 may be involved in post-stroke psychiatric changes related to anxiety and sociability.

Photothrombosis of corneal neovascularization by intravenous rose bengal and argon laser irradiation.

Management of corneal neovascularization by photocoagulation has been limited and rarely successful. We evaluated the efficacy and safety of the novel technique of photothrombosis to occlude corneal neovascularization. Sixteen rabbit corneas with previous ocular surface wounds that had healed with 360 degrees extensive neovascularization (persistent for 20 months) were used. After an intravenous injection of rose bengal solution (40 mg/kg of body weight [BW]), each vessel on the upper half of the cornea was occluded with a photochemically induced thrombus within ten shots of argon laser irradiation (514.5 nm, 130 mW, 63 microns, 0.2 s); those on the lower half were used as an internal control. Throughout the four-month study period, the treated vessels remained occluded, as evidenced by corneal fluorescein angiography. Corneal clarity was improved after treatment. A single injection of rose bengal at a dose of 8 mg/kg of BW or higher was sufficient for successful photothrombotic occlusion of corneal vessels within one hour of experimentation. Transient elevations of serum urea nitrogen, aspartate aminotransferase, alanine aminotransferase, alkaline phosphatase, and total bilirubin levels and decrease of serum phosphorus level were noted on the first day after injection with 40 mg/kg of BW of rose bengal solution.

Non-invasive induction of focal cerebral ischemia in mice by photothrombosis of cortical microvessels: characterization of inflammatory responses.

In this study, we adapted the original rat photothrombosis model of Watson et al. (Ann Neurol 17 (1985) 497) for use in mice by refining the application route of the dye, illumination and stereotactic parameters. After intraperitoneal injection of the photosensitive dye Rose bengal, subsequent focal illumination of the brain with a cold light source through the intact skull led to focal cortical infarcts of reproducible size, location and geometry. Cresyl violet histology displayed well-demarcated infarcts that matured with time in a predictable manner. Microglial responses, as assessed by immunocytochemistry, against F4/80 and CD11b antigens were rapid and complete at the infarct site, but delayed and incomplete in degenerating fiber tracts and ipsilateral thalamic nuclei. In contrast to the rat, where the expression of CD4 and CD8 antigens discriminate distinct subpopulations of lesion-associated phagocytes, the expression of both markers was low to absent in the mouse model. In both rats and mice, cerebral photothrombosis shares essential inflammatory responses with focal ischemia induced by middle cerebral artery occlusion. It may provide a useful model to study functional aspects of lesion-associated and remote molecular responses in transgenic mice.

Transient up-regulation of gamma-aminobutyric acid(A) receptor binding by lubeluzole after neocortical specify lesion in rats.

The effect of the neuroprotective drug lubeluzole on cortical receptor binding was investigated in animals with photothrombotic ischemic lesions. Control animals were treated with the inactive stereoisomer of the drug. Lubeluzole was applied intravenously as a single bolus (0.31 mg/kg) followed by a 1-h infusion of 0.31 mg/kg. Lubeluzole selectively increased gamma-amino-butyric acid(A) (GABA(A)) receptor binding but had no significant and/or consistent effects on N-methyl-D-aspartate (NMDA), (+/-)-alpha-amino-3-hydroxy-5-methylisoxazole-4-propionic acid (AMPA), and kainate receptors. Lubeluzole caused a significant up-regulation of GABA(A) receptor binding in the lesioned area as well as in unimpaired cortical areas of both hemispheres. This effect appeared in the hours following the lesion and peaked at 24 h. Our findings suggest that reduced cortical excitability brought about by increased binding capacities of GABA(A) receptors may contribute to the neuroprotective effect of lubeluzole.

Rose bengal induced corneal swelling: relation to inciting wavelength.

Cornea endothelial cells sensitized with rose bengal and exposed to light demonstrated a wavelength dependent alteration in physiological response. Corneas exposed to 549 millimicron swelled at rates similar to controls exposed to identical energy levels of incandescent light. Corneas exposed to 451, 500, 612, and 651 millimicron swelled at rates less rapid than corneas exposed to similar energy levels of incandescent light. The photodynamically induced physiological alteration of cornea endothelial cells approximately paralleled the spectral curve of rose bengal with its peak at 550 millimicron.

Still confused about rose bengal?

We have surveyed the ophthalmic literature of the last five years in an attempt to evaluate the use and usefulness of Rose Bengal staining as an aid to differential diagnosis in dry eyes. Included both as a criterion and as an adjunct measure of disease progression, Rose Bengal scores of patients with different dry eye conditions overlap, sometimes to a considerable extent. A mechanistic link between staining with this dye and disease etiology is unlikely; however, Rose Bengal could be a surrogate marker of changes in ocular surface physiology. The question whether the extent and pattern of staining with Rose Bengal provide the clinician with information not available from other tests, and in particular from fluorescein staining of the ocular surface, has to be answered positively, though the nature of this information is not clearly understood. A more widespread recognition that Rose Bengal is not a vital dye is necessary in order not to bias the interpretation of experimental results.

Changes in off-lesion endocochlear potential following localized lesion in the lateral wall.

Endocochlear potential (EP) was measured at various off-lesion sites after a small focal lesion was made in the lateral wall of the guinea pig cochlea. Lesions were produced by a photochemical reaction between systemically administered rose bengal and focused green light illumination. In 21 ears, continuous measurement for 30 min after onset of the reaction at turns apical or basal to the site of illumination revealed no significant changes in EP compared with the control value (p < 0.01). In another group of 43 ears, EP was measured at 3 days post-illumination. A significant decline was seen at every site located apical to the lesion (p < 0.001). Conversely, no significant change was measured at any site located basal to the lesion. These findings suggest that the decrease in EP assumes the form of a gradient from the lower to upper turns in the guinea pig cochlea.

[Experimental retinal vein occlusion induced by photodynamic approach].

OBJECTIVE: To develop a new photochemical method of experimental retinal vein occlusion and investigate the morphologic and histologic changes in the retina. METHODS: After intravenous injection of rose Bengal, the vessels next to the disc of miniature pigs in the experiment group (n = 15) were exposed to an endo-illuminator for 15 minutes. As a control group, the vessels next to the disc of each pig were exposed to the endo-illuminator without rose Bengal injection. After complete vascular occlusion, the eyes were observed at following times: one hour, and 1, 3, 7, 14, 21, and 28 days. After the 28th day, the eyes were enucleated and prepared for light and electron microscope examination. RESULTS: Histopathologic features were consistent with changes of retinal vein occlusion and formation of retinal vein thrombi was reliable. CONCLUSIONS: Photodynamic method combined with endo-illuminator was a simple, reliable and definitive experimental technique to produce retinal vein occlusion.

Unilateral infarction of the visual cortex (VC) induced an increase in dendritic spine turnover in contralateral VC.

After brain stroke, appropriate functional recovery is most important for improvement of quality of life. Cortical hemisphere contralateral to the infarction site plays an important role in functional recovery process. However, the underlying processes occurring in contralateral hemisphere during recovery has not yet been elucidated. We have previously reported that the turnover of synaptic spine of somatosensory cortex (SSC) is increased at 1st week after stroke in contralateral SSC infarction. After this period, neuronal circuit is remodeled, and functional compensation is achieved by processing bilateral information to remaining SSC [18]. In the present study, to examine whether similar changes are observed in different brain regions, we have induced an infarction in the visual cortex (VC). We found that the spinal remodeling in contralateral VC was also increased at 1st week after VC stroke. However, the magnitude of changes was not as great as those seen in SSC infraction. These results indicate that the regional difference may exist in the ability to induce functional recovery after ischemic brain damage.

Chemoablation of metastatic melanoma using intralesional Rose Bengal.

To study the effects of intralesional Rose Bengal for chemoablation of metastatic melanoma. Twenty-six target lesions in 11 patients with locoregionally recurrent disease were injected with the agent PV-10 (10% w/v Rose Bengal in saline) at a dose of 0.5 ml/cc lesion volume. An additional 28 untreated lesions were observed for potential bystander effect. The treatment was well tolerated and an objective response was observed in 12 target lesions, with an additional seven lesions remaining static over at least 12 weeks of observation. In this dose-evaluation study, response rate was dose dependent, increasing to 69% after higher dose injections. Nontarget lesions exhibited a 27% objective response rate that increased to 44% in patients exhibiting a positive response of target lesions. These findings indicate that intralesional Rose Bengal is nontoxic and could benefit patients with metastatic melanoma.

An assessment of the discomfort associated with the use of rose bengal 1% eyedrops on the normal human eye: a comparison with saline 0.9% and a topical ocular anaesthetic.

PURPOSE: To assess the time-dependent changes in ocular comfort following unilateral instillation of preservative-free rose bengal 1% eyedrops when compared with saline 0.9% or proxymetacaine 0.5%. METHODS: A total of 61 subjects, aged between 19 and 77 years, were asked to complete an ocular symptoms questionnaire, and then to indicate the comfort level for each eye on a 100-point visual analogue scale (VAS). A single drop of rose bengal was instilled in one eye (left or right) and a drop of saline or anaesthetic instilled in the other eye. The VAS assessments were repeated at an average of 4 and 7 min later. RESULTS: The instillation of rose bengal eyedrops produced an initial average reduction in comfort of 39.8 points, as compared with a reduction of 11.5 points following the anaesthetic and a reduction of 3.4 points for saline. However, the responses to rose bengal were highly variable, ranging from reductions of 1.5 to 84.6 points. In most subjects, some recovery had occurred within 6-8 min, but the comfort scores averaged 27.8 points below baseline levels prior to rose bengal. Slightly greater discomfort was noted by older subjects and those with darker irides. CONCLUSIONS: This study confirms that the use of rose bengal eyedrops can elicit a sensation of discomfort, but that this adverse reaction does not last very long. Based on the initial kinetics of recovery from discomfort, it is estimated that this should last no longer than 10-15 min (at least for those without significant ocular surface disease).