Correlative microscopy and block-face imaging (CoMBI): a 3D imaging method with wide applicability in the field of biological science.

Correlative microscopy and block-face imaging (CoMBI) is an imaging method, which is characterized by the ability to obtain both serial block-face images as a 3-dimentional (3D) dataset and sections for 2-dimentional (2D) light microscopic analysis. These 3D and 2D morphological data can be correlated with each other to facilitate data interpretation. CoMBI is an easy-to-install and low-cost 3D imaging method since its system can be assembled by the researcher using a regular microtome, consumer digital camera, and some self-made devices, and its installation and instruction manuals are open-source. After the first release of CoMBI method from our laboratory, CoMBI systems have been installed in more than a dozen laboratories and are used for 3D analysis of various biological specimens. Typical application of CoMBI is 3D anatomical analysis using the natural color and contrast of the specimen. We have been using CoMBI for analyzing human brain to obtain the fine 3D anatomy as a reference to determine the causes of neurological diseases and to improve the effectiveness of surgery. Recently, we have been using CoMBI for detecting the colors of chromogens, which are used for labeling specific molecules. Mouse embryos colored with X-gal, a conventional chromogen for detecting LacZ products, were imaged using CoMBI, and the 3D distribution of X-gal was successfully visualized. Thus, CoMBI can now be used for many purposes, including 3D anatomical analysis, 2D microscopy using sections, and 3D distribution of specific molecules. These suggest that CoMBI should be more widely used in the field of biological research.

Heavy ion irradiation induces autophagy in irradiated C2C12 myoblasts and their bystander cells.

Autophagy is one of the major processes involved in the degradation of intracellular materials. Here, we examined the potential impact of heavy ion irradiation on the induction of autophagy in irradiated C2C12 mouse myoblasts and their non-targeted bystander cells. In irradiated cells, ultrastructural analysis revealed the accumulation of autophagic structures at various stages of autophagy (i.e. phagophores, autophagosomes and autolysosomes) within 20 min after irradiation. Multivesicular bodies (MVBs) and autolysosomes containing MVBs (amphisomes) were also observed. Heavy ion irradiation increased the staining of microtubule-associated protein 1 light chain 3 and LysoTracker Red (LTR). Such enhanced staining was suppressed by an autophagy inhibitor 3-methyladenine. In addition to irradiated cells, bystander cells were also positive with LTR staining. Altogether, these results suggest that heavy ion irradiation induces autophagy not only in irradiated myoblasts but also in their bystander cells.

Insufficient membrane fusion in dysferlin-deficient muscle fibers after heavy-ion irradiation.

Recently, SJL/J mice have been used as an animal model in studies of dysferlinopathy, a spectrum of muscle diseases caused by defects in dysferlin protein. In this study we irradiated muscle fibers isolated from skeletal muscle of SJL/J mice with heavy-ion microbeam, and the ultrastructural changes were observed by electron microscopy. The plasma membrane of heavy-ion beam irradiated areas showed irregular protrusions and invaginations. Disruption of sarcomeric structures and the enhancement of autophagy were also observed. In addition, many vesicles of varying size and shape were seen to be accumulated just beneath the plasma membrane. This finding further supports the recent hypothesis that dysferlin functions as a membrane fusion protein in the wound healing system of plasma membrane, and that the defect in dysferlin causes insufficient membrane fusion resulting in accumulation of vesicles.

Heavy ion microbeam irradiation induces ultrastructural changes in isolated single fibers of skeletal muscle.

The effects of heavy ion microbeams on muscle fibers isolated from mouse skeletal muscles were examined by electron microscopy. The plasma membranes of heavy ion beam-irradiated areas of muscle fibers showed irregular protrusions and invaginations. In the cytoplasm, an irregular distribution of microfilaments was found near the plasma membrane. Sarcoplasmic reticula in the irradiated regions showed a distended appearance with flocculent material within the lumen. These changes were seen as early as 2 min after irradiation, and persisted until as late as 22 min after irradiation. Many autophagic vacuoles could be seen at 7 min after irradiation. At 22 min, the vacuoles became more prominent and showed more variety. These observations suggest that heavy ion beam irradiation causes disruption of the cellular architecture and the autophagy is involved in removal of this disruption.

Histological effect and protein expression in subthreshold transpupillary thermotherapy in rabbit eyes.

OBJECTIVE: To investigate the histological effect of subthreshold transpupillary thermotherapy (TTT) on the retina. METHODS: We performed TTT in normal pigmented rabbit eyes using an 810-nm diode laser with spot size of 1.2 mm, power of 50 mW, and varying durations of 15, 30, or 60 seconds. Four weeks later, fluorescein angiography was performed, and the enucleated eyes were examined by means of electron microscopy and immunohistochemical staining. RESULTS: Funduscopy immediately and at 4 weeks showed no discernable changes at TTT sites, and fluorescein angiography at 4 weeks showed no abnormalities. However, electron microscopy showed photoreceptor and retinal pigment epithelium cell disruption, changes more prominent with longer durations of treatment. Immunohistochemical staining was positive for heat shock protein 60, heat shock protein 70, tumor necrosis factor alpha, and vascular cell adhesion molecule 1 in the photoreceptors and retinal pigment epithelium at TTT sites. Untreated control eyes showed no staining. CONCLUSIONS: Despite the absence of changes evident by funduscopy and fluorescein angiography, TTT resulted in dose-dependent histological changes in photoreceptors and retinal pigment epithelium. The induction of heat shock proteins, cytokines, and cell adhesion molecules may play a role in the tissue response to subthreshold TTT. Clinical Relevance Unrecognized damage to the retina and retinal pigment epithelium may contribute to visual loss in eyes that undergo subthreshold TTT.

Changing patterns of intraocular inflammatory disease in Japan.

PURPOSE: We investigated the frequencies and clinical characteristics of Japanese patients with uveitis. METHODS: Records of 189 patients referred from April 1999 to March 2001 were retrospectively reviewed. RESULTS: Fifty-six patients (29.6%) had anterior uveitis, 13 (6.9%) intermediate uveitis, 59 (31.2%) posterior uveitis, 58 (30.7%) panuveitis, and three (1.6%) papillitis. The most common diagnoses were Vogt-Koyanagi-Harada (VKH) disease (10.1%), biopsy-proven or presumed sarcoidosis (9.5%), acute anterior uveitis (7.9%), tuberculosis (6.9%), and Behçet's disease (5.8%). Seventy-three patients (38.6%) were treated with local therapy alone, and 95 patients (50.3%) required systemic therapy. Ocular complications developed in 19.6% of patients, and systemic complications in 2.1%. CONCLUSIONS: These results confirm a continued high frequency of VKH disease and sarcoidosis, but suggest a decreased frequency of Behçet's disease and an increased frequency of tuberculosis. Roughly one-half of the patients required systemic treatment in addition to local therapy, and ocular and/or systemic complications developed in one-fifth of the patients.

Bilateral anterior optic neuritis in adult measles infection without encephalomyelitis.

PURPOSE: To describe anterior optic neuritis in adult measles infection. DESIGN: Interventional case report. METHODS: A 31-year-old woman presented with bilateral visual loss 6 days after the onset of maculopapular rash. Complete ophthalmic and neurologic examinations, radiologic studies, and lumbar puncture were performed. RESULTS: Visual acuities were counting fingers in both eyes, with bilateral optic disk hyperemia and swelling noted. Neurologic examination was unremarkable, and computed tomography and magnetic resonance imaging of the brain were normal. The cerebrospinal fluid (CSF) was devoid of white cells, although measles immunoglobulin M (IgM) antibodies were detected in both CSF and serum. Intravenous corticosteroids were administered, and clinical findings resolved within 1 month. A fall in serum IgM and a rise in serum IgG titers were observed. CONCLUSIONS: Although rare, optic neuritis in the absence of encephalomyelitis may occur in measles. Whether treatment is effective is unknown.

Tuberculin skin testing in uveitis patients and treatment of presumed intraocular tuberculosis in Japan.

PURPOSE: To evaluate the results of tuberculin skin testing in Japanese patients with intraocular inflammation and to assess the outcome of treatment for presumed intraocular tuberculosis in selected patients. DESIGN: Prospective, noncomparative, interventional case series. PARTICIPANTS: One hundred twenty-six patients, newly referred to the Ocular Inflammation Service at the Kyorin Eye Center from April 1998 to August 2000, underwent systemic evaluation for the diagnosis and/or treatment of uveitis. METHODS: Tuberculin skin testing with purified protein derivative was performed as part of the systemic evaluation. The diagnosis of presumed intraocular tuberculosis was made when findings were consistent with possible intraocular tuberculosis, the tuberculin skin test was positive (induration more than 10 mm), and no other cause of uveitis was suggested by symptoms, signs, or ancillary testing. Using these criteria, 10 patients were given a diagnosis of presumed intraocular tuberculosis and treated with antituberculosis therapy consisting of isoniazid, with or without rifampicin. Some of these patients also received a tapered course of oral corticosteroids after the initiation of antituberculosis treatment. None of the patients had any signs or symptoms of acquired immunodeficiency syndrome. MAIN OUTCOME MEASURES: Visual acuity and ophthalmologic examination to assess degree of intraocular inflammation. RESULTS: Twenty-six of the 126 patients (20.6%) had a positive tuberculin skin test result. Ten of these 26 patients (38.5%) were treated for a diagnosis of presumed intraocular tuberculosis. Nine patients had no evidence of pulmonary tuberculosis, and one patient had presumed tuberculous hilar lymphadenitis. The predominant clinical finding was choroidal or optic disc nodule in three patients, retinal vasculitis in three patients, and choroiditis in four patients. Nine patients exhibited decreased intraocular inflammation with treatment. CONCLUSIONS: Roughly one fifth of the uveitis patients who underwent systemic evaluation had a positive tuberculin skin test result, and 9 of 10 selected skin test-positive patients with clinical findings consistent with intraocular tuberculosis had a favorable response to antituberculosis therapy. These results suggest that intraocular tuberculosis continues to be a major diagnostic consideration for uveitis patients in Japan.