Taketoshi Wakabayashi

Up-regulation of Hrk, a regulator of cell death, in retinal ganglion cells of axotomized rat retina.

Hrk, a regulator of cell death, belongs to the family of pro-apoptotic BH3-only proteins and is known to induce apoptosis in nerve tissue. We examined Hrk gene expression to clarify the role of BH3-only proteins in apoptosis of retinal ganglion cells (RGCs) after optic nerve transection in the adult rat. Reverse transcription-polymerase chain reaction showed that Hrk was up-regulated in retina from 12 h after axotomy, and continued to be elevated for 1 week. In situ hybridization histochemistry revealed that Hrk was expressed in a sub-population of axotomized RGCs. These results suggest that Hrk is involved in the induction of apoptosis in RGCs after optic nerve transection.

Survival of axotomized retinal ganglion cells in peripheral nerve-grafted ferrets

PURPOSEnPeripheral nerve (PN) grafting to the optic nerve stump stimulates not only axonal regeneration of the axotomized retinal ganglion cells (RGCs) into the grafted PN but also their survival. The purpose of the present study was to determine the number, distribution, and soma diameter of only surviving RGCs without regenerated axons and surviving RGCs with regenerated axons in PN-grafted mammals.nnnMETHODSnA segment of PN was grafted to the optic nerve stump of adult ferrets. Two months after the PN grafting, surviving RGCs with regenerated axons were retrogradely labeled with granular blue (GB) and stained with RGC-specific antibody C38. Surviving RGCs without regenerated axons were identified as C38-positive cells without GB labeling.nnnRESULTSnTwenty-one percent of RGCs survived axotomy after PN grafting in the area centralis (AC), whereas 47% survived in the peripheral retina. Twenty-six percent of surviving RGCs in the AC exhibited axonal regeneration, which was higher than that in the peripheral retina. Soma diameter histograms revealed that RGCs with regenerated axons showing both GB and C38 positivity were in the large soma diameter ranges. In contrast, the soma diameter distribution of surviving RGCs that did not have regenerated axons showed a peak in the smaller soma diameter ranges.nnnCONCLUSIONSnThe present data suggest that PN grafting promotes survival of axotomized RGCs more effectively in the peripheral retina than in the AC. Among surviving RGCs, the larger cells exhibited axonal regeneration into the grafted PN, whereas the axons of smaller cells did not to regenerate in either the AC or the peripheral retina.

Rapid and sensitive diagnosis of adenoviral keratoconjunctivitis by loop-mediated isothermal amplification (LAMP) method

Purpose. To develop a new method to detect and type adenoviruses directly from conjunctival scrapings using loop-mediated isothermal amplification (LAMP) with adenovirus (ad) type specific primer. Methods. Using primers specific for the gene of ad1, ad3, ad4, ad8, ad19 and ad37, heat denatured adenovirus DNA was amplified by the LAMP and polymerase chain reaction (PCR). Alkaline lysed adenovirus prototype and conjunctival scrapings were also used directly as templates. Results. Type specific primers amplified ad genes of the corresponding ad prototype specifically. The specific amplification was observed in both heat denatured and alkaline lysed samples. The amplified product was first detected within 45 min. Ad genotypes of clinical samples determined by the LAMP method were almost identical to those determined using the PCR-sequencing method. Conclusions. LAMP based isothermal amplification of adenovirus genome for detection and typing of adenoviruses is faster than PCR based methods. This new method will be useful for rapid diagnosis and typing of adenoviral conjunctivitis.

Monoclonal Antibody C38 Recognizes Retinal Ganglion Cells in Cats and Rats

We developed monoclonal antibody C38 which specifically recognizes retinal ganglion cells (RGCs) in flatmount preparations of cat and rat retinas. We first induced immunological tolerance in Balb/c mice against axotomized rat retinas which lack most of the RGCs. Then the mice were immunized with intact rat retinas to produce antibodies against RGCs. Monoclonal antibody C38 appeared to be specific for cat RGCs based on immunoreactivities seen in flatmounts and vertical sections of the retina. In rats, we verified that over 90% of retrogradely labeled RGCs were immunoreactive for C38 antibody. In axotomized rat retinas, surviving RGCs were labeled with C38 without erroneous labeling of glial cells. The antigen that C38 recognized was 24 kDa in molecular weight and found in cerebrum, cerebellum, and spinal cord as well as retina. It is suggested that monoclonal antibody C38 is a useful label for RGCs.

JNK inhibitory kinase is up-regulated in retinal ganglion cells after axotomy and enhances BimEL expression level in neuronal cells

Optic nerve transection results in retinal ganglion cell (RGC) death in adult mammals, after the alteration of gene expression of RGCs. To elucidate the molecular mechanism by which axotomy induces RGC death, we isolated the molecules up‐regulated after optic nerve transection. One of these, axomtomy‐related gene (ARG)357, an 895‐amino‐acid protein containing a complete serine–threonine kinase domain, was isolated from a subtraction library of the rat retina. The sequence showed that this gene was a rat homolog of human c‐Jun N‐terminal kinase (JNK) inhibitory kinase and so belonged to the germinal center kinase‐VIII subfamily of Sterile20s protein kinase. We designated ARG357 as rat JNK inhibitory kinase (JIK). Rat JIK was expressed ubiquitously in various tissues and was highly expressed in the retina, with selective expression in RGCs. After axotomy, BimEL and Hrk, which are BH3‐only proteins, and rat JIK were up‐regulated in RGCs. Overexpression of rat JIK in neuronal cells up‐regulated the expression of BimEL, but not that of Hrk. These results indicate that JIK may contribute to axotomy‐induced RGC death by up‐regulating the expression of BH3‐only protein.

Posterior capsule staining and posterior continuous curvilinear capsulorhexis in congenital cataract

We report 2 cases of indocyanine green (ICG) staining used for posterior continuous curvilinear capsulorhexis (PCCC) in congenital cataract combined with anterior vitrectomy. In the first case, because of corneal opacity, the visibility of the posterior capsule was poor without staining. After the extraction of the cataract, a PCCC was performed after ICG staining of the posterior capsule. In the second case, after cataract removal, ICG staining was used to better visualize the posterior capsule. In both cases, the PCCC was successfully completed because of better visualization of the stained posterior capsule flap against the transparent anterior hyaloid face of the vitreous. Clear visual axes have been maintained.

Monoclonal antibody C38 labels surviving retinal ganglion cells after peripheral nerve graft in axotomized rat retina.

C38 is a monoclonal antibody that labels retinal ganglion cells in both intact and axotomized rat retinas. We report here that C38 labeled retinal ganglion cells that survived after optic nerve section and peripheral nerve graft in rats. Furthermore, with combination of the retrograde labeling, we succeeded to distinguish surviving retinal ganglion cells without axonal regeneration from those with regenerating axon.

Comparison of Micafungin and Fluconazole for Experimental Candida Keratitis in Rabbits

Purpose: To evaluate the efficacy of subconjunctival injection of micafungin in the treatment of experimental Candida albicans keratitis in rabbits compared with fluconazole. Methods: In 1 eye of 24 New Zealand white rabbits, C. albicans (5 × 104 yeast cells) was inoculated in the corneal stroma. The animals were randomly assigned to 3 groups and received subconjunctival injection of 0.5 mL of 0.1% micafungin, 0.2% fluconazole, or physiologic saline once a day for 3 weeks. The eyes were examined slit-lamp biomicroscopically and histopathologically. The clinical course of fungal keratitis was compared among the 3 groups. In another 36 rabbits, a microbiological examination was performed using a quantitative isolate recovery technique, and the numbers of colony-forming units were compared among groups. Results: The clinical scores were significantly lower in the micafungin group than in the other 2 groups throughout the study period (P < 0.0001 ∼ P = 0.0027, Bonferroni multiple comparison). The fluconazole group showed significantly lower clinical scores than the saline group on day 18 (P = 0.0343). At the end of the study period, there were significant differences between the saline and micafungin groups (P < 0.0001), the saline and fluconazole groups (P = 0.0072), and the fluconazole and micafungin groups (P = 0.0013). Histopathologically, similar results were obtained. Moreover, the results of the microbiological examination nearly matched the clinical and histopathologic findings. Conclusions: Subconjunctival administration of micafungin was effective in the treatment of experimental Candida keratitis. Local application of micafungin to the eye would be a feasible treatment option for clinical fungal keratitis.

Heat Treatment Enhances Healing Process of Experimental Pseudomonas Corneal Ulcer

We investigated the effects of hyperthermia on the healing process of experimental Pseudomonas corneal ulceration (PCU). Hartley guinea pigs were used to develop animal models of PCU. As a heat source, disposable chemical pocket warmers were applied. The healing process of PCU was compared between the heat-treated corneas and the control corneas. The severity of infection and the degree of angiogenesis were classified by a clinical scoring system. The animals were euthanized 14 days after infection and the corneas were submitted for histopathological examination. The expression of vascular endothelial growth factor (VEGF) was examined immunohistochemically. Comparative reverse transcription polymerase chain reaction was performed to measure the expression level of VEGF in the cornea. Hyperthermia significantly promoted corneal epithelization and neovascularization in the PCU model. Heat treatment significantly decreased the number of viable Pseudomonas organisms present in PCU. On immunohistochemistry, the heated cornea demonstrated more intense staining for VEGF. Comparative reverse transcription polymerase chain reaction showed upregulation of the expression level of VEGF mRNA in the heat-treated cornea. Hyperthermia accelerated the healing process of PCU with increased corneal neovascularization. Angiogenesis may play an important role in the PCU healing process, which is enhanced by the heat treatment.

Detection of CMV DNA in the aqueous humor of AIDS patients with CMV retinitis by AMPLICOR CMV test.

The present study was performed to detect the cytomegalovirus (CMV) DNA in the aqueous humor from the eyes of acquired immunodeficiency syndrome (AIDS) patients with CMV retinitis. Detection of CMV DNA in the aqueous humor in the eyes with active CMV retinitis was compared with detection of CMV DNA in inactive retinitis. CMV DNA in the aqueous humor was evaluated before and after treatment with intravitreal injection of ganciclovir. CMV DNA in the aqueous humor was measured by AMPLICOR CMV test. Forty-two eyes of 35 AIDS patients were diagnosed ophthalmoscopically as having CMV retinitis that was subclassified as either active or inactive. The active and inactive CMV retinitis cases were distinguished based on clinical evaluations and fundus photographs. The results showed that 37 of the 42 eyes (88.1%) were positive for CMV DNA prior to treatment, while in 29 of these 37 eyes (78.4%), the aqueous humor became CMV DNA-negative after the treatment. Successful treatment with the intravitreal injection of ganciclovir was associated with a reduction in the detection of CMV DNA in the aqueous humor. CMV DNA was not detected in the aqueous humor of patients with quies cent CMV retinitis. In conclusion, the AMPLICOR CMV test was found to be a reliable tool for differentiating active and inactive CMV retinitis, and is useful for helping to select the optimal treatment regimen. The intravitreal injection of ganciclovir is highly effective in reducing detectable CMV DNA in the aqueous humor.