Early Ultrastructural Changes Research Articles

Monitoring Cutaneous Leishmaniasis Lesions in Mice Undergoing Topical Miltefosine Treatment

A study was conducted on BALB/c mice infected with Leishmania (Leishmania) amazonensis to analyse the effects of 0.5% miltefosine (MTF) hydrogel treatment on cutaneous leishmaniasis (CL) lesions. The mice were treated for 25 days topically, and lesion sizes, parasite loads, histopathology, ultrastructure, cytokines including interleukin 4 (IL-4), tumour necrosis factor alfa (TNFα), interferon gamma (IFNγ), IL-10, and vascular endothelial growth factor (VEGF) profiles were evaluated on days 0, 12, 25, and 85. After 12 days of treatment, the lesion sizes and parasite numbers decreased. By day 60 post treatment, there were no lesions and only a few parasites. At day 25, there was a temporary papillomatosis reaction, an increase in mast cells, a few giant cells, and granulomas, and a decrease in diffuse inflammatory infiltrate and parasites. Transmission electron microscopy (TEM) examination showed early ultrastructural changes, including macrophages without parasites and vacuoles containing electrodense material. At the different evaluated times, the cytokine regulation indexes (ICRs) decreased for IL-4, TNFα, and VEGF. According to the study, the 0.5% MTF hydrogel was effective and showed positive results from the early stages of usage. The MTF directly targeted parasites, downregulated the release of IL-4, TNFα, and VEGF, increased mast cell production, and induced granuloma reaction during evaluation periods.

Early Ultrastructural Changes in Biopsies From Patients With Symptomatic CKD of Uncertain Etiology

Although the investigation of chronic kidney disease of uncertain etiology (CKDu) has identified many possible influencing factors in recent years, the exact pathomechanism of this disease remains unclear. In this study, we collected 13 renal biopsies from patients with symptomatic CKDu (Sym-CKDu) from Sri Lanka with well-documented clinical and socioeconomic factors. We performed light microscopy and electron microscopic evaluation for ultrastructural analysis, which was compared with 100 biopsies from German patients with 20 different kidney diseases. Of the 13 Sri Lankan patients, 12 were men (92.3%), frequently employed in agriculture (50%), and experienced symptoms such as feeling feverish (83.3%), dysuria (83.3%), and arthralgia (66.6%). Light microscopic evaluation using activity and chronicity score revealed that cases represented early stages of CKDu except for 2 biopsies, which showed additional signs of diabetes. Most glomeruli showed only mild changes, such as podocyte foot process effacement on electron microscopy. We found a spectrum of early tubulointerstitial changes including partial loss of brush border in proximal tubules, detachment of tubular cells, enlarged vacuoles, and mitochondrial swelling associated with loss of cristae and dysmorphic lysosomes with electron-dense aggregates. None of these changes occurred exclusively in Sym-CKDu; however, they were significantly more frequent in these cases than in the control cohort. In conclusion, our findings confirm the predominant and early alterations of tubular structure in CKDu that can occur without significant glomerular changes. The ultrastructural changes do not provide concrete evidence of the cause of CKDu but were significantly more frequent in Sym-CKDu than in the controls.

Early cellular development induced by ecdysteroid in sex-specific wing degeneration of the wingless female winter moth.

The winter moth, Nyssiodes lefuarius, exhibits striking sexual dimorphism in wing form; males have functional wings of normal size, whereas females lack wings. We previously found that the steroid hormone 20-hydroxyecdysone (20E) triggered massive programmed cell death (PCD) only in the female pupal wing epithelium; however, when and how early sexual trait development of the pupal wings is initiated during pupal-adult metamorphosis remains obscure. To clarify the detailed morphological changes and mechanisms underlying early sexual trait development and cell death, we examined the effects of 20E on early ultrastructural and histological changes in the pupal wing epithelium of both sexes. Before the onset of adult differentiation, no morphological differences were observed in the epithelial cells of both sexes at an ultrastructural level. When 5.4µg of 20E was injected into pupae of both sexes at 15days after the onset of pupation, retraction of the wing epithelium from the pupal cuticle was initiated at day 2 after 20E injection in both sexes. Although overt degeneration of wing tissue was not still obvious, apoptotic body-like structures and auto-phagosomes were visible at day 3 after 20E injection in females, whereas development of scale precursor cells started on day 4 after injection in males. Our results suggest that (1) the injection of 20E induced sexually dimorphic changes in the pattern of organelle distribution in wing epithelial cells, and (2) abnormally shaped mitochondria in the cytoplasm of the female wing epithelium might be involved in the PCD that occurs during wing tissue degeneration.

Characterisation of early ultrastructural changes in the cerebral white matter of CADASIL small vessel disease using high-pressure freezing/freeze-substitution.

The objective of this study was to elucidate the early white matter changes in CADASIL small vessel disease. We used high-pressure freezing and freeze substitution (HPF/FS) in combination with high-resolution electron microscopy (EM), immunohistochemistry and confocal microscopy of brain specimens from control and CADASIL (TgNotch3R169C ) mice aged 4-15months to study white matter lesions in the corpus callosum. We first optimised the HPF/FS protocol in which samples were chemically prefixed, frozen in a sample carrier filled with 20% polyvinylpyrrolidone and freeze-substituted in a cocktail of tannic acid, osmium tetroxide and uranyl acetate dissolved in acetone. EM analysis showed that CADASIL mice exhibit significant splitting of myelin layers and enlargement of the inner tongue of small calibre axons from the age of 6months, then vesiculation of the inner tongue and myelin sheath thinning at 15months of age. Immunohistochemistry revealed an increased number of oligodendrocyte precursor cells, although only in older mice, but no reduction in the number of mature oligodendrocytes at any age. The number of Iba1 positive microglial cells was increased in older but not in younger CADASIL mice, but the number of activated microglial cells (Iba1 and CD68 positive) was unchanged at any age. We conclude that early WM lesions in CADASIL affect first and foremost the myelin sheath and the inner tongue, suggestive of a primary myelin injury. We propose that those defects are consistent with a hypoxic/ischaemic mechanism.

Early histological and ultrastructural changes in expanded murine scalp

ABSTRACT Tissue expansion has been widely used for plastic, reconstructive, and esthetic surgeries. A mouse scalp expansion model can effectively mimic the characteristics of human skin expansion. However, a detailed study of the histological features and ultrastructural characteristics of expanded scalp is lacking, especially early ultrastructural changes. Here, a mouse scalp expansion model was established and the expanded scalp samples were obtained on day 2 (group I) and 4 (group II) post final injection. Histological analysis revealed epidermal thickening, dermal thinning, subcutaneous fat thinning, and capsule formation in the expanded samples. Ultrastructural evaluation showed the presence of keratinocytes with numerous tonofibrils and damaged mitochondria, and several ruptured collagen fibers and increased number of active fibroblasts and myofibroblasts were observed in the dermis and capsules. Adipocyte dedifferentiation was detected in the expanded samples of both groups, but formation of autophagosomes was only detected in the dermal fibroblasts of group I. Thus, early changes in expanded tissue should be carefully monitored, as it may help avoid dermal thinning and promote expanded tissue regeneration.

Ultrastructural Changes in Lung Allografts

Introduction Antibody mediated rejection (AMR) is a well-recognised cause of allograft injury; however only recently there is a growing recognition in lung transplantation (LTx). Allograft dysfunction can be multifactorial and challenges with C4d stain interpretation and the lack of specificity of histological criteria account for some of the difficulties in diagnosing pulmonary AMR. Donor Specific Antibodies (DSA) target the endothelium. In fact, in renal transplantation, endothelial activation markers and early ultrastructural changes of the endothelium correlate with AMR. The aim of this study was to identify ultrastructural changes in the endothelium of lung allografts in the presence of DSA, with the intent of determining if prospective use of electron microscopy might be helpful in establishing a diagnosis of AMR in LTx recipients. Materials & Methods The LTx database was queried for cases of pulmonary AMR between 07/2009 & 09/2015. The ISHLT guidelines were used to categorise these cases into definite, probable & possible AMR. Retrospective ultrastructural studies were performed on all these biopsies- either fresh or paraffin embedded. Cases of acute cellular rejection (4) and normal lung (3) were evaluated as controls. The findings on ultrastructural examination were correlated with the presence of DSA & diagnosis of AMR using the Pearson’s chi-square test of statistical significance. Results & Discussion Eleven patients (male, 54.5%) were included in this study.Mean age was 53yrs. Three cases were combined lung & heart Tx. We identified 7 cases of AMR (1 definite, 1 probable & 5 possible). The median time from LTx to biopsy (bx) was 210 days, with median follow up of 1063 days. Bronchiolitis Obliterans Syndrome was diagnosed in 5 patients. Ultrastructural studies were performed on 10 AMR bxs (pt 6:2 bxs; pt 7:3 bxs; all other pts: 1bx each), 4 ACR bxs & 4 controls. The results are shown.The presence of DSA as well as the ISHLT diagnosis of AMR significantly correlated with endothelial swelling, cytoplasmic vacuolisation & septal edema (p<0.01; each). Septal infiltration by neutrophils was only identified in AMR cases, although it did not achieve statistical significance. Importantly, these differences were still significant when using resin or paraffin based samples for EM, allowing larger retrospective studies to be performed; although endothelial swelling & vacuolisation occurred in non-DSA paraffin samples with EM.Conclusion Donor specific anti-HLA antibodies are associated with endothelial injury of the allograft. Ultrastructural findings such as endothelial swelling & vacuolization help in diagnosing antibody mediated allograft injury. Although paraffin embedded biopsy material can be used for this purpose, it is not without limitations and larger prospective studies are needed to validate these findings. The effect of DSA on the endothelial pathways will be important in all solid organ transplantation.

Pectoral Vessel Density and Early Ultrastructural Changes in Broiler Chicken Wooden Breast Myopathy

In wooden breast myopathy (WBM) of broiler chickens, the pectoralis major muscles show abnormally hard consistency and microscopical myodegeneration of unknown aetiology. To date, previous studies have focused primarily on chronic WBM and ultrastructural descriptions of early WBM are lacking. The aim of this study was to elucidate the pathogenesis of WBM by light microscopical morphometry of vessel density and the ultrastructural description of early WBM changes with transmission electron microscopy. The pectoral vessel density was compared between unaffected chickens (n=14) and two areas of focal WBM in affected chickens (n=14). The transverse myofibre area per vessel was highest in the unaffected area of muscle from cases of focal WBM, significantly higher (P=0.01) than in macroscopically unaffected tissue, indicating that relatively decreased blood supply may trigger the development of WBM. The ultrastructural study included unaffected chickens (n=3), two areas of focal WBM from affected chickens (n=3) and areas of diffuse WBM from affected chickens (n=3). The morphologically least affected myofibres within the WBM lesion areas in light microscopy exhibited ultrastructural changes of increased sarcoplasmic reticulum diameter and mitochondrial hyperplasia. Such changes originate typically from osmotic imbalance, for which the most likely aetiologies in WBM include tissue hypoxia or myodegeneration of the surrounding myofibres. The findings suggest that a relative reduction of blood supply in the major pectoral muscle occurs in the early phase of WBM, which may be linked to the ultrastructural changes of osmotic imbalance.

The short-term effects of C-peptide on the early diabetes-related ultrastructural changes to the podocyte slit diaphragm of glomeruli in rats.

This study aimed to investigate the structural changes in the slit diaphragm, caused by early diabetes, and the nephroprotective effect of C-peptide. Streptozotocin-induced type 1 diabetic Wistar rats were divided into control, control plus C-peptide, early diabetes, and early diabetes plus C-peptide groups. C-peptide was infused into rats for one day. The slit diaphragm component proteins podocin, CD2AP, and nephrin, were stained immunofluorescently and their distribution quantitatively analyzed by determining the overlapping area ratio. The interfoot process gap length was measured from electron microscopic images. Diabetic duration correlated best with the area ratio of podocin and CD2AP (r = 0.626), followed by other protein combinations, showing progressive change in the slit diaphragm structure. C-peptide-treatment did not alter area ratios. The interfoot process gap length was wider in diabetic rats (p < 0.05) and did not narrow with C-peptide-treatment in either control or diabetic rats (both p < 0.05). Diabetes widened the interfoot process gap length and distorted the slit diaphragm structure progressively and heterogeneously in rats with early diabetes; this was not altered by C-peptide-treatment. The nephroprotective effect of C-peptide in decreasing the glomerular filtration rate appears to be functional rather than structural.

Early muscle and brain ultrastructural changes in polymerase gamma 1‐related encephalomyopathy

Mutations affecting the mitochondrial DNA-polymerase gamma 1 (POLG1) gene have been shown to cause Alpers-Huttenlocher disease. Ultrastructural data on brain and muscle tissue are rare. We report on ultrastructural changes in brain and muscle tissue of two sisters who were compound heterozygous for the c.2243G>C and c.1879C>T POLG1 mutations. Patient 1 (16 years) presented with epilepsia partialis continua that did not respond to antiepileptic treatment. Neuroimaging showed right occipital and bithalamic changes. Light microscopy from a brain biopsy performed after 3 weeks suggested chronic encephalitis showing astro- and microgliosis as well as perivascular CD8-positive T-cells. However, immunosuppressive therapy failed to improve her condition. When her 17-year-old sister (patient 2) also developed epilepsy, an intensified search for metabolic diseases led to the diagnosis. On electron microscopy mitochondrial abnormalities mainly affecting neurons were detected in the brain biopsy of patient 1, including an increase in number and size, structural changes and globoid inclusions. In patient 2, light and electron microscopy on a muscle biopsy confirmed a mitochondrial myopathy, also revealing an increase in mitochondrial size and number, as well as globoid inclusions. Neurons may be the primary target of mitochondrial dysfunction in brains of patients with Alpers disease related to POLG1 mutations. During early disease stages, brain histopathology may be misleading, showing reactive inflammatory changes.

Childhood and adolescent obesity in cardiorenal metabolic syndrome and Type 2 diabetes: a clinical vignette and ultrastructure study

SUMMARY Childhood–adolescent overweight and obesity (CAOO) have grown to pandemic proportions during the past decade. The younger onset of obesity will have dire future complications manifesting as earlier onset end-organ disease in a disproportionate number of younger adults. This clinical snapshot begins with a clinical vignette of a 13 year old female with obesity, atherogenic lipid profile, hypertension with early urinary changes of chronic kidney disease and prediabetes. We have focused on the early ultrastructural changes found in two rodent models of obesity (Zucker obese [fa/fa] rat model of obesity, insulin resistance and prediabetes and db/db mouse model of obesity, insulin resistance and Type 2 diabetes mellitus) in their adolescent ages. Our focus is on remodeling in the obese adipose tissue, emphasizing the omental depot, the skeletal muscle, the islet and β cells and the end-organs including the myocardial and renal tissues. Obesity seems to be the driving force behind this process occurring...

Early Ultrastructural Changes in Renal Allografts: Correlation With Antibody-Mediated Rejection and Transplant Glomerulopathy

Transplant glomerulopathy (TG) is associated with antibody-mediated renal allograft rejection (AMR) and reduced graft survival. Histologically, TG is typically seen >1 year posttransplantation. However, ultrastructural changes including glomerular endothelial swelling, subendothelial widening and early glomerular basement membrane duplication are associated with development of TG but appear much earlier. We examined the specificity of these changes for AMR, and whether these are inevitably associated with development of TG. Of 98 for cause renal allograft biopsies carried out within 3 months of transplantation with available serologic data, 17 showed C4d-positive AMR and 16 had histologic changes of AMR and donor-specific antibodies (DSA), but no C4d. All three ultrastructural changes were seen in 11 of 17 biopsies with C4d-positive AMR, 8 of 16 with histologic changes of AMR and DSA but no C4d, and 0 of 65 without histologic changes of AMR and/or DSA (p < 0.0001 for both of the former groups vs. the latter). Twenty patients with positive DSA (18 with histologic changes of AMR and 11 C4d-positive) had ≥1 follow-up biopsy; eight developed overt TG 3.5-30 months posttransplantation. Among the 18 patients with DSA and histologic changes of AMR, 11 C4d-positive and 7 C4d-negative, treatment for AMR after the early biopsy significantly reduced subsequent development of overt TG.

High Glucose-induced Altered Basement Membrane Composition and Structure Increases Trans-endothelial Permeability: Implications for Diabetic Retinopathy

Purpose: The following study was designed to investigate early biosynthetic and ultrastructural changes that alter functional properties of the basement membrane (BM) and affect vascular permeability in diabetic retinopathy.Materials and Methods: To determine whether altered matrix synthesis affects cell monolayer permeability, rat retinal endothelial cells (RRECs) were grown for 4 days to confluency in normal (N, 5 mM) or high glucose (HG, 30 mM) medium on transwell inserts and subjected to an in vitro cell monolayer permeability assay. Inserts were cut out and viewed under a transmission electron microscope to assess extracellular matrix (ECM) accumulation and cell morphology. In parallel cell cultures, fibronectin and collagen IV protein expression were determined using Western Blot analysis.Results: Electron microscopic analysis of cells exposed to short-term HG showed no difference in inter-endothelial cell tight junctions (TJs) or in the number of vesicles or coated pits compared to those of normal cells. However, ECM accumulation underlying HG cells was significantly increased compared to that of cells grown in N medium (139 ± 7% of control, p = 0.04), with areas of focal thickening. Western blot analysis showed increased fibronectin and collagen IV expression (152 ± 24% of control, p = 0.01; 146 ± 16% of control, p = 0.02, respectively) in cells grown in HG compared to those grown in N medium. Cell monolayers grown in HG exhibited increased permeability to FITC-dextran compared to cells grown in N medium (134 ± 15% of control, p = 0.02).Conclusions: High glucose-induced excess ECM accumulation and altered composition underlies structural and functional changes that allow increased permeability. This finding provides evidence for the first time that the thickened vascular basement membrane contributes to the development of excess permeability seen in diabetic retinopathy.

Cerivastatin induces type-I fiber-, not type-II fiber-, predominant muscular toxicity in the young male F344 rats

3-hydroxy-3-methylglutaryl coenzyme A reductase inhibitors (statins) are associated with adverse skeletal muscle toxicity, but the underlying mechanism remains unclear. To investigate the pathological mechanism of statin-induced myotoxicity, cerivastatin (20 ppm; corresponding to 2 mg/kg/day) was dietarily administered to young male F344 rats for 10 days, and time-course clinical observations, measurement of plasma creatine kinase activity, and light and electron microscopy of type I fiber-predominant skeletal muscle (soleus) or type II fiber-predominant skeletal muscles (extensor digitorum longus and tibialis anterior), were performed. Clinical symptoms including weakness of hind limbs, staggering gait and body weight loss, accompanied by marked plasma creatinine kinase elevation in rats fed cerivastatin at around Day 6 to 8. Interestingly, microscopic examination revealed that cerivastatin-induced muscle damages characterized by hypercontraction (opaque) and necrosis of the fibers were of particular abundance in the soleus muscle at Day 8, whereas these histological lesions in the extensor digitorum longus and tibialis anterior were negligible, even at Day 9. Prior to manifestation of muscle damage, swollen mitochondria and autophagic vacuoles in the soleus were observed as the earliest ultra structural changes at Day 6; then activated lysosomes, disarray of myofibril and dilated sarcoplasmic reticulum vesicles became ubiquitous at Day 8. These results demonstrate that cerivastatin induces type I fiber-predominant muscles injury, which is associated with mitochondrial damage, in young male F344 rats. Since the rat exhibiting type I fiber-targeted injury is a unique animal model for statin-induced myotoxicity, it will be useful for gaining insight into mechanisms of statin-induced myotoxicity.

Permeability, Ultrastructural Changes, and Distribution of Novel Proteins in the Glomerular Barrier in Early Puromycin Aminonucleoside Nephrosis

Background/Aims: It is still unclear what happens in the glomerulus when proteinuria starts. Using puromycin aminonucleoside nephrosis (PAN) rats, we studied early ultrastructural and permeability changes in relation to the expression of the podocyte-associated molecules nephrin, α-actinin, dendrin, and plekhh2, the last two of which were only recently discovered in podocytes. Methods: Using immune stainings, semiquantitative measurement was performed under the electron microscope. Permeability was assessed using isolated kidney perfusion with tracers. Possible effects of ACE inhibition were tested. Results: By day 2, some patchy foot process effacement, but no proteinuria, appeared. The amount of nephrin was reduced in both diseased and normal areas. The other proteins showed few changes, which were limited to diseased areas. By day 4, foot process effacement was complete and proteinuria appeared in parallel with signs of size barrier damage. Nephrin decreased further, while dendrin and plekhh2 also decreased but α-actinin remained unchanged. ACE inhibition had no significant protective effect. Conclusions: PAN glomeruli already showed significant pathology by day 4, despite relatively mild proteinuria. This was preceded by altered nephrin expression, supporting its pivotal role in podocyte morphology. The novel proteins dendrin and plekhh2 were both reduced, suggesting roles in PAN, whereas α-actinin was unchanged.

Ultrastructural Changes in the Ileum of White BALB/C Mice Experimentally Infected with Salmonella hadar

Problem statement: Recently, Salmonella hadar has been isolated and identified from goat in Iraq. The purpose of the present study was to in vestigate the ultrastructural changes in the ileum epithelial cells of mice experimentally infected wi th Salmonella hadar . Approach: The white BALB/c mice inoculated orally with Salmonella enterica serovar hadar strain and their ileums were examined by transmission electron microscopy at 24, 48, 72, 96 and 120 h after inoculation. Results: The challenge organism appeared adhered to the mucosal microvilli of the ileum from 24 h post- inoculation. The early ultrastructural changes char acterized by local derangement with slight swelling of the proximal ends of the microvilli. In addition , there was ruffling of the apical cell surfaces of the microvilli with cytoplasmic vacuolization of entero cytes due to invading by the organisms. The Challenge organism was usually intact and enclosed by a membrane. The damage of the epithelial cells started as a minor lesion at 24 h and became severe after 48, 72 and 96 h post infection. The more sev ere ultrastructural changes in the ileum occurred at 12 0 h post infection, which revealed hypertrophy of goblet cell, dilatation of endoplasmic reticulum, s evere cytoplasmic vacuolization, thickening of the nuclear membrane and several Salmonella Containing Vacuoles (SCV). Conclusion: Our results revealed that the cellular invasion by Salmonella hadar may occurred early than 24 h because of the obvious alterations seen in the cells of the ileum at this time of infection. In addition, the continu ation of cellular pathological changes for 120 h post inf ection may refers that S. hadar has some mechanisms which aid it to survive and replicate wi thin intestinal cells. We recommend further studies with early observation periods in order to determin e the invasion time.

Cryosurgery: Early Ultrastructural Changes in Liver Tissue In Vivo

Experimental observations with regard to freezing in vitro cell lines and fluid systems led to the application of low temperatures to in vivo biological systems. For the first time, this report describes the cryosurgical response of liver parenchyma and the early ultrastructural cellular changes in liver tissue, i.e., cryosurgery, in vivo. Forty-eight animals were used for the experiment. The dogs were divided into four groups. In group A, the liver tissue was frozen to -80 degrees C and in group B, to -180 degrees C. Temperatures of -80 degrees C and -180 degrees C in contact with liver tissue was selected for cryosurgical exposure. For transmission electron microscopy, the specimens were taken immediately and 1 h after the finishing of the freeze-thaw cycles intraoperatively. Further, the next specimens were taken in 24 h, this time also intraoperatively. The electronic microscopic analysis showed that, after local cryodestruction at temperatures of -80 degrees C and -180 degrees C, similar processes occurred within the liver tissue in the early postcryosurgical phase-immediately and 1 h after cryosurgical session. The hepatocytes in the center of the cryozone changed upon thawing. Ultrastructural changes in the hepatic cells, where the first signs of dystrophic processes had been noticed, were increased. Our new insights prove on the cell level that suddenly and progressively damaged liver cells in the postcryosurgical zone lead to aseptic cryoaponecrosis and then to aseptic cryoapoptosis of vital normal tissue. The vascular capillary changes and circulatory stagnation demonstrate together with cryoaponecrosis and cryoapoptosis the anti-angiogenesis mechanisms, which are some of the main mechanisms of biological tissue injury following the low temperature exposure.

Neuroprotective effects of high-dose vs low-dose melatonin after blunt sciatic nerve injury

Melatonin, the secretory product of the pineal gland, has potent antioxidant properties. The aim of this study was to compare the effects of low-dose (10 mg/kg) vs high-dose (50 mg/kg) melatonin on early lipid peroxidation levels and ultrastructural changes in experimental blunt sciatic nerve injury (SNI). We believe this to be the first study to assess the dose-dependent neuroprotective effects of melatonin after a blunt peripheral nerve injury. Rats were randomly allocated into 5 groups of 10 animals each. The SNI only rats underwent a nerve injury procedure. The SNI plus vehicle group received SNI and intraperitoneal injection of vehicle (diluted ethanol) as a placebo. The SNI plus low-dose or high-dose melatonin groups received intraperitoneal melatonin at doses of 10 mg/kg or 50 mg/kg, respectively. Controls had no operation, melatonin or vehicle injection. SNI was induced by clamping the sciatic nerve at the upper border of the quadratus femoris for 2 min. Sciatic nerve samples were harvested 6 h after nerve injury and processed for biochemical and ultrastructural analysis. Trauma increased the lipid peroxidation of the sciatic nerve by 3.6-fold (153.85 +/- 18.73 in SNI only vs 41.73 +/- 2.23 in control rats, P < 0.01). Low (P = 0.02) and high (P < 0.01) doses of melatonin attenuated the nerve lipid peroxidation by 25% and 57.25%, respectively (65.76 +/- 2.47 in high-dose vs 115.08 +/- 7.03 in low-dose melatonin groups). Although low-dose melatonin reduced trauma-induced myelin breakdown and axonal changes in the sciatic nerve, high-dose melatonin almost entirely neutralized any ultrastructural changes. Our results suggest that melatonin, especially at a dose of 50 mg/kg, has a potent neuroprotective effect and can preserve peripheral neural fibers from lipid peroxidative damage after blunt trauma. With further investigations, we hope that these data may prove useful to clinicians who treat patients with nerve injuries.

β-Amyloid-induced Dynamin 1 Degradation Is Mediated by N-Methyl-D-Aspartate Receptors in Hippocampal Neurons

Alzheimer disease (AD) is a progressive, neurodegenerative disorder that leads to debilitating cognitive deficits. Although little is known about the early functional or ultrastructural changes associated with AD, it has been proposed that a stage of synaptic dysfunction might precede neurodegeneration in the development of this disease. Unfortunately, the molecular mechanisms that underlie such synaptic dysfunction remain largely unknown. Recently we have shown that beta-amyloid (Abeta), the main component of senile plaques, induced a significant decrease in dynamin 1, a protein that plays a critical role in synaptic vesicle recycling, and hence, in the signaling properties of the synapse. We report here that this dynamin 1 degradation was the result of calpain activation induced by the sustained calcium influx mediated by N-methyl-D-aspartate receptors in hippocampal neurons. In addition, our results showed that soluble oligomeric Abeta, and not fibrillar Abeta, was responsible for this sustained calcium influx, calpain activation, and dynamin 1 degradation. Considering the importance of dynamin 1 to synaptic function, these data suggest that Abeta soluble oligomers might catalyze a stage of synaptic dysfunction that precedes synapse loss and neurodegeneration. These data also highlight the calpain system as a novel therapeutic target for early stage AD intervention.

Early and late immunohistochemical and ultrastructural changes associated with functional impairment of the lachrymal gland following external beam radiation

The aim of this study was to investigate scintigraphic, immunohistological and ultrastructural changes associated with radiation-induced dysfunction of the lachrymal gland in an established experimental animal model. Ten rabbits were randomized into two groups and used for the study; in the control as well as experimental group, the Schirmer-test, lachrymal gland scintigraphy, and immunohistological and ultrastructural investigations were carried out prior to irradiation and 72 h as well as 1 month after single-dose irradiation with 15 Gy. Seventy-two hours after irradiation, secretion reduction evaluated by the Schirmer-test was evident. At this phase, we could observe a decrease in the expression of alpha-SMA and a re-distribution of tenascin-C matrix. Ultrastructural changes of acinar and myoepithelial cells were noticed; simultaneously, disturbance in the primary (99m)TcO(4)- uptake as well as significant reduction of the lachrymal ejection fraction was assessed scintigraphically. These changes were still evident 1 month following irradiation but became less intensive. Single-dose irradiation with 15 Gy implicates a functional impairment of the lachrymal gland, which is associated with early immunohistological and ultrastructural alterations. These changes may represent objective surrogate parameters for radiogenic dysfunction and prerequisites for further investigations on radioprotection of lachrymal glands during radiotherapy of the periorbital region.

CYP2A5-MEDIATED ACTIVATION AND EARLY ULTRASTRUCTURAL CHANGES IN THE OLFACTORY MUCOSA: STUDIES ON 2,6-DICHLOROPHENYL METHYLSULFONE

2,6-Dichlorophenyl methylsulfone (2,6-diClPh-MeSO2) is a potent olfactory toxicant reported to induce endoplasmic reticulum (ER) stress, caspase activation, and extensive cell death in mice. The aim of the present study was to examine cytochrome P450 (P450)-dependent bioactivation, nonprotein sulfhydryl (NP-SH) levels, and early ultrastructural changes in mouse olfactory mucosa following an i.p. injection of 2,6-diClPh-MeSO2 (32 mg/kg). A high covalent binding of 2,6-diClPh-14C-MeSO2 in olfactory mucosa S9 fraction was observed, and the CYP2A5/CYP2G1 substrates coumarin and dichlobenil significantly decreased the binding, whereas the CYP2E1 substrate chlorzoxazone had no effects. An increased bioactivation was detected in liver microsomes of mice pretreated with pyrazole, known to induce CYP2A4, 2A5, 2E1, and 2J, and addition of chlorzoxazone reduced this binding. 2,6-DiClPh-14C-MeSO2 showed a marked covalent binding to microsomes of recombinant yeast cells expressing mouse CYP2A5 or human CYP2A6 compared with wild type. One and 4 h after a single injection of 2,6-diClPh-MeSO2, the NP-SH levels in the olfactory mucosa were significantly reduced compared with control, whereas there was no change in the liver. Ultrastructural studies revealed that ER, mitochondria, and secretory granules in nonneuronal cells were early targets 1 h after injection. We propose that lesions induced by 2,6-diClPh-MeSO2 in the mouse olfactory mucosa were initiated by a P450-mediated bioactivation in the Bowman's glands and depletion of NP-SH levels, leading to disruption of ion homeostasis, organelle swelling, and cell death. The high expression of CYP2A5 in the olfactory mucosa is suggested to play a key role for the tissue-specific toxicity induced by 2,6-diClPh-MeSO2.