Swelling Of Rough Endoplasmic Reticulum Research Articles

Impacts of ingested MWCNT-Embedded nanocomposites in Japanese medaka (Oryzias latipes)

Polymer nanocomposites combine the versatile, lightweight characteristics of polymers with the properties of nanomaterials. Polyethylene terephthalate glycol (PETG) is commonly used in polymer additive manufacturing due to its controllable transparency, high modulus, and mechanical properties. Multi-walled carbon nanotubes (MWCNTs) add tensile strength, electrical conductivity, and thermal stability. The increased use of nanocomposites has led to concern over potential human health risks. We assessed morphologic alterations to determine impacts of ingested abraded nanocomposites compared to its component materials, pristine MWCNTs (1000 mg/L) and PETG. Adult transparent Japanese medaka (Oryzias latipes) were administered materials via oral gavage in 7 doses over 16 days. In vivo observations revealed altered livers and gallbladders following exposure to pristine MWCNTs and nanocomposites. Subsequent histologic sections showed fish exposed to pristine MWCNTs had highly altered biliary structures, and exposure to nanocomposites resulted in hepatocellular alteration. Thyroid follicle proliferation was also observed in fish exposed to materials containing MWCNTs. Transmission electron microscopy of livers showed that hepatocytes of fish exposed to MWCNTs had widespread swelling of rough endoplasmic reticulum, pronounced lysosomal activity, and swelling of intrahepatic biliary passageways. Fish exposed to nanocomposites had areas of degenerated hepatocytes with interspersed cellular debris. Each analysis showed that fish exposed to pristine PETG were most similar to controls. These results suggest that MWCNTs are the source of toxicity in abraded nanocomposite materials but that nanocomposites may also have some unique effects. The similarities of many teleost and mammalian tissues are such that these findings may indicate human health risks.

Role of dietary selenium in alleviating bisphenol A toxicity of liver albino rats: Histological, ultrastructural, and biomarker assessments.

Bisphenol A (BPA) is used as a plasticizer in polycarbonate plastics. It has been used in consumer products and epoxy resins for decades as protective coatings and linings for food and beverage bottles. This can trigger human reactions to BPA which interferes with estrogen receptors. Our study explored the ameliorative effects of selenium (Se) in male rats on liver damage caused by BPA. Rats were divided into four groups at random: The first one obtained olive oil and acted as a control. Se (0.5mg/kg diet) was given for the second group. The third one was treated with BPA (10mg/kg body weight/day) orally. Concomitantly Se (0.5mg/kg diet) and BPA (10mg/kg body weight/day) were given orally in the fourth one. Liver specimens were prepared for light, electron microscopes and the serum samples were screened for biochemical markers. In the BPA received group, histological findings indicated apoptotic hepatic histological changes such as sinusoidal congestion, cytoplasmic vacuolation and leukocyte infiltration. Ultrastructurally, the same group had mitochondrial degeneration, rough endoplasmic reticulum swelling, and nuclear pyknosis, as well as fat droplet deposition and lysosome enhancement. Liver enzymes: In the BPA group, alanine aminotransferase (ALT) and aspartate aminotransferase (AST) have been substantially increased. Moreover, histological and ultrastructural improvements were seen in the rat population treated with BPA and Se, whereas ALT and AST levels were lowered and malondialdehyde (MDA), glutathione peroxidase (GPx), human C reactive protein (hCRP), and the serum levels of interleukin-6 (IL-6) were significantly modulated. PRACTICAL APPLICATIONS: Bisphenol A (BPA) is used in the manufacturing of polycarbonate plastic (e.g., water bottles, baby bottles) and epoxy resins (e.g., inner coating in metallic food cans). It is a non-polymer preservative for other plastics, one of the contaminants of the atmosphere and a common endocrine estrogenic disruptor. Our study explored the ameliorative effects of selenium (Se) in male rats on liver damage caused by BPA. Rats were divided into four groups at random: The first one obtained olive oil and acted as a control. Se (0.5mg/kg diet) was given for the second group. The third one was treated with BPA (10mg/kg body weight/day) orally. Concomitant Se (0.5mg/kg diet) and BPA (10mg/kg body weight/day) were given in the fourth one. Liver specimens were prepared for light, electron microscopes and the serum samples were screened for biochemical markers.

Astragaloside protects rat brain from microwave-induced functional injuries via restoring acetylcholine and normalizing electroencephalogram.

Studies from our group and others have reported that 30mW/cm2 microwave could damage the structures of rat hippocampus, as well as impair the neuronal functions. The neuroprotective effects of astragaloside, purified from Astragalus membranaceus, have been demonstrated in animal models of neurodegenerative diseases. In this study, we found that 30mW/cm2 microwave impaired spatial learning and memory ability in rats, while astragaloside could significantly alleviate the injuries. The pathological analysis also showed that astragaloside protected neurons from microwave-induced damages, such as mitochondrial swelling and cavitation, rough endoplasmic reticulum swelling and dilation, synaptic gap disappearing, and vesicle aggregation. Moreover, microwave-induced structural damage of synapse resulted in downregulation of acetylcholine, an important neurotransmitter for information transmission, while astragaloside could protect the structure of synapse, as well as restore the acetylcholine level in rat hippocampus. Furthermore, astragaloside also accelerated the recovery of brain electroencephalogram (EEG) after microwave exposure, indicating that astragaloside could promote the normalization of neuronal functions. In conclusion, astragaloside protected the morphological structures and restored acetylcholine level in rat hippocampus, which could improve brain functions via normalizing brain EEG. Therefore, astragaloside might be a promising candidate to treat microwave-induced injuries of central nervous system (CNS).

Tetracycline uptake by pak choi grown on contaminated soils and its toxicity in human liver cell line HL-7702

Tetracycline (TC) can enter the human body via the soil-vegetable-human food chain; therefore, it is necessary to understand the toxicity of TC to humans through vegetables grown on contaminated soils. The present study combined an enzyme-linked immunosorbent assay method and an HL-7702 cell model and assessed the bioavailability and toxicity of TC from pak choi (Brassica campestris L. ssp. chinensis) grown on TC-contaminated soils. The results showed that the degradation rate of TC in black soil was significantly higher than that in purplish clay, while the results for TC uptake in pak choi were opposite. The bioaccessibility of TC was found to be higher in pak choi grown on purplish clay (5.67–7.59%) than in that grown on black soil (5.22–6.77%). It is suggested that soil properties contribute to the uptake of TC by pak choi. More fertile soil contained lower TC concentrations and thus mediated lower TC toxicity to humans. It may seem comforting that TC concentrations in the edible parts of pak choi are often found to be below safe limits. However, the TC diagnosis method showed that even moderate increases in TC concentrations in pak choi may induce oxidative stress, liver injury, mitochondrial cristae and rough endoplasmic reticulum swelling, and early apoptosis in liver cells HL-7702. The pak choi grown in purplish clay showed higher TC cytotoxicity than that grown in black soil. The TC cytotoxicity of raw pak choi was found to be higher than that of cooked pak choi. These results provide direct evidence of effective ways to prevent TC toxicity in humans.

Radiation-induced damage to lacrimal glands: an ultrastructural study in Sprague Dawley rats

ABSTRACTInjury to lacrimal glands represents a major health problem after radiation therapy of the head and neck malignancies. Accordingly, this study aimed to investigate significant ultrastructural changes of lacrimal glands and some of their underlying mechanisms following the exposure to different fractionated doses of irradiation. In this study, 28 Sprague Dawley (SD) rats were assigned to four groups (seven rats each): Group I acted as control and received no irradiation. Groups II–IV received fractionated irradiation of 5 Gy (100 cGy/fraction daily for 5 days), 9 Gy (300 cGy/fraction daily for 3 days), and 20 Gy (one fraction), respectively. One month after the experiment, examination of lacrimal glands with transmission electron microscopy (TEM) demonstrated dose-dependent ultrastructural changes in the lacrimal acinar and intralobular ductal epithelial cells. In the acinar cells, there were swollen rough endoplasmic reticulum, irregularly shaped nuclei with chromatin condensation, mitochondrial damage, and retention of secretory granules. Intaralobular ductal epithelial cells showed loss of surface microvilli and damage to mitochondria. In addition to the potential direct effects of irradiation on lacrimal acinar and intralobular ductal epithelial cells, damage to blood vessels and nerve endings seemed to mediate some of the underlying mechanisms of these irradiation-induced ultrastructural changes. In conclusion, using TEM reveals that lacrimal gland is highly sensitive to even small doses of irradiation therapy; in addition, swelling of rough endoplasmic reticulum and aberrant nuclei are the most encountered structural changes. Damage to blood vessels and nerve endings might mediate some of the underlying mechanisms of irradiation-induced secondary injury in lacrimal glands.

Evaluation of allowable time and histopathological changes in warm ischemia of the uterus in cynomolgus monkey as a model for uterus transplantation.

The objective of this study was to examine the allowable warm ischemic time and pathological changes due to ischemia and reperfusion injury in the uterus of the cynomolgus monkey as a model for uterus transplantation. Six female cynomolgus monkeys were used in the study. The uterus was resected from the vaginal canal and connected through the bilateral ovarian and uterine arteries and veins only. One animal was used as a control. In the other five animals, the bilateral uterine and ovarian vessels were clamped for 0.5, 1, 2, 4 and 8h, respectively. Biopsy of the smooth muscle tissue of corpus uteri was performed after each ischemic time and after subsequent reperfusion for 3h. Biopsy samples were observed by light and electron microscopy. Menstruation recovery was monitored. There were no particular findings in both light and electron microscopy after ischemia for up to 2h and after subsequent reperfusion. There were no marked changes after ischemia for 4h, but dilated nuclear pores and rough endoplasmic reticulum swelling were found after reperfusion. These changes also occurred, along with mitochondrial swelling and cristae loss after ischemia for 8h, and plasma membrane loss, nuclear fragmentation and chromatin condensation were found after reperfusion. Periodical menstruation restarted in all animals with ischemia up to 4h, but the animal with ischemia for 8h had amenorrhea and uterine atrophy. The uterus of the cynomolgus monkey tolerates warm ischemia for up to 4h.

Morphological effects of neem (Azadirachta indica A. Juss) seed oil with known azadirachtin concentrations on the oocytes of semi-engorged Rhipicephalus sanguineus ticks (Acari: Ixodidae).

The concern about the harmful effects caused by synthetic pesticides has led to the search for safe and ecological alternatives for pest control. In this context, the neem tree (Azadirachta indica) stands out due to its repellent properties and effects on various arthropods, including ticks. For this reason, this study aimed to demonstrate the potential of neem as a control method for Rhipicephalus sanguineus ticks, important vectors of diseases in the veterinary point of view. For this, R. sanguineus semi-engorged females were subjected to treatment with neem seed oil enriched with azadirachtin, its main compound, and ovaries were assessed by means of morphological techniques in conventional light microscopy, confocal laser scanning microscopy, and transmission electron microscopy. Neem demonstrated a clear dose-dependent effect in the analyzed samples. The observed oocytes presented, especially in the groups treated with higher concentrations of neem oil, obvious signs of cytoplasmic disorganization, cellular vacuolization, nuclear and nucleolar irregularity, dilation in mitochondrial cristae, alterations in mitochondrial matrix, and swelling of rough endoplasmic reticulum. Intracellular microorganisms were observed in all analyzed groups, reinforcing the importance of ticks in the transmission of pathogens. A greater quantity of microorganisms was noted as the concentration of neem increased, indicating that the damaged oocytes may be more susceptible for their development. Such morphological alterations may promote future damages in reproductive performance of these animals and demonstrate the potential of neem seed oil for the control of R. sanguineus ticks, paving the way for new, cheaper, and safer methods of control.

Ultrastructural and antioxidative changes in lupine embryo axes in response to salt stress

Embryo axes of lupine (<em>Lupinus luteus</em> L. ‘Mister’) were subjected to 0.1 M NaCl salt stress for 24 and 48 h. The ultrastructure modification and adjustment of antioxidant enzymes activities and izoenzymes profiles were observed. In cells of lupine embryo axes grown for 48 hours in medium with 0.1 M NaCl mitochondria took the forked shape and bulges of the outer mitochondrial membranes appeared. Moreover, the inflating and swelling of rough endoplasmic reticulum (RER) lumen and fragmentation of RER were noticed. The level of H<sub>2</sub>O<sub>2</sub> was higher in salt treated embryo axes after 24 hours and increase of thiobarbituric acid reactive substances was observed after both 24 and 48 h of salt treatment. Native gel electrophoresis showed increased intensities of bands for catalase isozymes in response to salt stress, whereas activity of catalase was higher only in embryo axes grown for 48 h in control conditions. Appearance of two new isoforms of ascorbate peroxidase was observed after 48 h only under control condition, however increased activities were stated for both control and salt-stress condition after 48 h. No changes in isozymes pattern for superoxide dismutase were observed, but significant decrease in superoxide dismutase activity was noticed in relation to time and salt stress. Possible role of these enzymes in salt stress tolerance is discussed. The 0.1 M salt stress is regarded as a middle stress for lupine embryo axes and the efficiency of stress prevention mechanisms is proposed.

Proteomic identification of hippocampal proteins vulnerable to oxidative stress in excitotoxin-induced acute neuronal injury

Excitotoxicity is involved in seizure-induced acute neuronal death, hypoxic–ischemic encephalopathy, and chronic neurodegenerative conditions such as Alzheimer's disease. Although oxidative stress has been implicated in excitotoxicity, the target proteins of oxidative damage during the course of excitotoxic cell death are still unclear. In the present study, we performed 2D-oxyblot analysis and mass spectrometric amino acid sequencing to identify proteins that were vulnerable to oxidative damage in the rat hippocampus during kainic acid (KA)-induced status epilepticus. We first investigated the time course in which oxidative protein damage occurred using immunohistochemistry. Carbonylated proteins, a manifestation of protein oxidation, were detected in hippocampal neurons as early as 3h after KA administration. Immunoreactivity for 8-hydroxy-2′-deoxyguanosine (8-OHdG) was also elevated at the same time point. The increase in oxidative damage to proteins and DNA occurred concomitantly with the early morphological changes in KA-treated rat hippocampus, i.e., changes in chromatin distribution and swelling of rough endoplasmic reticulum and mitochondria, which preceded the appearance of morphological features of neuronal death such as pyknotic nuclei and hypereosinophilic cytoplasm. Proteomic analysis revealed that several hippocampal proteins were consistently carbonylated at this time point, including heat shock 70kDa protein 4, valosin-containing protein, mitochondrial inner membrane protein (mitofilin), α-internexin, and tyrosine 3-monooxygenase/tryptophan 5-monooxygenase activation protein (14-3-3 protein). We propose that oxidative damage to these proteins may be one of the upstream events in the molecular pathway leading to excitotoxic cell death in KA-treated rat hippocampus, and these proteins may be targets of therapeutic intervention for seizure-induced neuronal death.

Effects of human contraceptive on reproduction and offspring in Chrysomya megacephala

Objective To investigate the effect of human contraceptive (HC) as ability to suppress the reproductive success of blow fly, Chrysomya megacephala (Fabricius) ( C. megacephala) and offspring under controlled laboratory conditions. Methods Adult C. megacephala were fed with low (0.036 mg/mL) and high dose (0.072 mg/mL) HC (Microgest®, Thailand), containing levonorgestrel and ethinyl estradiol, in their drinking water for 7 days. Three experiments were set; experiment I with fed only in parental males, experiment II with fed only in parental females and experiment III with fed in both males and females. All experiments were then maintained for 3 generations after crossing and inbreeding. Results A lower ovariole production and less fully mature ovarioles were evident in F1, F2 and F3 than control when parent males, females and both had been fed with high dose HC. Cellular changes during spermatogenesis in F1, F2 and F3 testes was confirmed using transmission electron microscopy (TEM), showing the low level of condensed chromatin, necrotic chromatin, irregularities and degenerated nuclear envelope in the nucleus. In the cytoplasm, mitochondrial swelling, rough endoplasmic reticulum swelling as well as vacuolated cytoplasm were noticed. As for the sperm per se, we found the degenerated nuclei and/or incomplete mitochondrial derivative, axoneme and vacuolated flagella. Regarding deformity in F1, F2 and F3 ovariole, ultrastructural alteration observed by scanning electron microscopy (SEM) included malformations involving fragile enveloping peritoneal sheath, cracked ovarioles, peel away chorion, crumbled eggshell and incomplete development; whereas TEM presented malformed and disorganized mass of cells, proteic yolk granules and vacuolated vesicles. Conclusions Administer of HC to adult C. megacephala caused ovariole reduction, less matured ovariole and affected cellular changes in testes and ovariole of offspring up to F3.

Diabetogenic effects of FK506 on renal subcapsular islet isografts in rat

Previously we demonstrated prevention of immune rejection in rat islet allografts by continuous subcutaneous (s.c.) administration of FK506 and also showed that FK506 might have diabetogenic effects (Ryu and Yasunami (1991) Transplantation, 52, 599–605). The purpose of the present study was to characterize further diabetogenic effects of FK506 on renal subcapsular islet isografts in rat. Continuous s.c. administration of FK506 (3 mg/kg/day) for 35 days produced glucose intolerance in the recipients as demonstrated by intravenous (i.v.) glucose tolerance test at the end (35 days) and after discontinuation (90 days) of FK506 administration. Morphologically, beta cells in the grafts of FK506-treated group were degranulated at 35 and 120 days after transplantation. Electron microscopically, degranulation, marked swelling of rough endoplasmic reticulum, Golgi apparatus and mitochondria were detected in beta cells of the grafts treated with FK506 at 35 days, and at 120 days there was moderate structural recovery in the organella. These findings clearly demonstrate that FK506 has diabetogenic effects on renal subcapsular islet isografts in rat and also suggests potential reversibility of damages by FK506 in beta cells of the grafts.

Fine structure and detoxification kinetics in kupffer cells after injection of endotoxin in rats

The aim of this study was to clarify the role of Kupffer cells in the mechanism of endotoxin-induced liver injury. The study on fine structure of Kupffer cells was performed after the injection of endotoxin. The endotoxin(Escherichia coli lipopolysaccharide 026 : B6. 1.5mg/100 g of body weight) was intraperitoneally injected in Sprague-Dewley rats. Animals were sacrificed at 1/4, 1/2, 1, 2, 4, 8, 16, 24, 72 and 120 hours after the injection of endotoxin. Livers were extirpated and processed to be examined by light and electron microscopy. The results obtained were summerized as follows: Early changes observed in liver after endotoxin injection included the increased number and hypertrophy of Kupffer cells, infiltration of neutrophils and presence of fibrin thrombi within the sinusoids. The continuous increase of the Kupffer cells in number with hypertrophy, congestion and infiltration of inflammatory cells within the sinusoids were observed. Hepatocytes showed fatty change and occasional necrosis. At 72 hours the congestion decreased. At 120 hours the number of Kupffer cells was increased, but the morphology of Kupffer cells became similar to that of the control group. The numbers and sizes of primary and secondary lysosomes and amount of euchromatin of Kupffer cells increased. Swellings and increase in number of mitochondria, Golgi complex, smooth endoplasmic reticulum, rough endoplasmic reticulum were evident. Microthrombi were present within the sinusoids. The swelling of rough endoplasmic reticulum and mitochondria, decrease of glycogen particles, fatty change, hypoxic vacuoles, pyknotic nuclei and occasional necrosis were observed in hepatocytes. At 72 hours the number of secondary lysosomes in Kupffer cells decreased. At 120 hours the morphology of Kupffer cells became similar to that of the control group. According to these results, it was postulated that the endotoxin was initially taken up by pinocytosis into Kupffer cells and degraded in secondary lysosomes of activated Kupffer cells. Kupffer cells may play an important role in the defense mechanism of liver during endotoxemia. The dysfunction of Kupffer cells and ischemia by sinusoidal microthrombi may cause liver injury.

Growth inhibition and morphological changes of LLC-PK 1 induced bt ultimobranchial calcitonins

Ultimobranchial calcitonins (CTs), known to stimulate cAMP production, inhibited the growth of a porcine kidney cell line LLC-PK 1. This inhibition was accompanied by degenerative changes including vacuole formation and cell detachment. The electron microscopic study revealed marked swelling of rough endoplasmic reticulum (RER). Other cAMP-increasing agents such as human CT, arginine, vasopressin and forskolin showed less growth inhibitory activities and no induction of the degenerative changes. These results indicate that the growth inhibition of LLC-PK 1 by ultimobranchial CTs is mainly due to cellular death caused by the swelling of RER via a signalling pathway other than the cAMP-dependent event(s).

Ofloxacin in juvenile non-human primates and rats. Arthropathia and drug plasma concentrations

Arthropathia in juvenile animals is the most important toxic effect induced by quinolones. We conducted pharmacokinetic and morphological studies with ofloxacin on non-human primates (Callithrix jacchus, Marmosets) and rats. In the marmoset, electron microscopy and the application of immuno-morphological methods proved to be suitable for the detection of specific alterations in cartilage (e.g. loss of proteoglycans and altered chondrocytes). Subsequently performed electron microscopic examinations in rats showed similar specific alterations of the femur cartilage surface after multiple oral applications of 600 mg ofloxacin/kg body wt. These results were correlated with pharmacokinetic data obtained for the same species. After single oral application of 100, 300 or 600 mg ofloxacin/kg body wt to 5 week-old rats peak plasma levels were achieved 15-45 min after administration indicating a rapid absorption of the drug. The following peak concentrations were measured for the three doses applied (mean +/- SD): 8.9 +/- 2.1, 22.6 +/- 7.5 mg/l and 33.5 +/- 9.8 mg/l, respectively. After 360 min the concentrations were 1.1 +/- 0.4, 5.9 +/- 2.5 and 15.9 +/- 5.1 mg/l, respectively. After subcutaneous injection of 100 mg ofloxacin/kg body wt the mean peak concentration was 27.7 +/- 2.6 mg/l after 45 min (0.5 +/- 0.2 mg/l after 360 min). In the marmoset higher plasma concentrations were measured with comparable doses. One, 3, and 6 h after the last of nine administrations of 200 mg ofloxacin/kg body wt, the mean (+/- SD) plasma concentrations were: 42.7 +/- 16.7, 40.6 +/- 9.5, and 26.5 +/- 3.6 mg ofloxacin/l plasma. Typical alterations of the joint cartilage of juvenile rats (e.g. opened chondrocyte cavities, swelling of rough endoplasmic reticulum and mitochondrial swelling in the chondrocytes) were induced by oral administration of ofloxacin at doses that were approximately 100 times higher than therapeutic ones, but led to peak plasma concentrations which were only approximately 10 times above the therapeutic level.

Ultrastructure of experimental cocaine hepatotoxicity.

Cocaine is a potent hepatotoxin in mice. It is converted in the liver by a minor oxidative pathway to the active metabolite, norcocaine nitroxide. Previous studies have shown evidence of a lipid peroxidative mechanism of toxicity, including increased conjugated diene absorption by hepatic microsomal lipids following a single 60 mg per kg i.p. dose of cocaine in DBA/2Ha mice. To explore this mechanism further, morphologic changes in the livers of DBA/2Ha mice were examined following the same dose of cocaine. The first ultrastructural change seen was dilatation of rough endoplasmic reticulum in centrilobular hepatocytes 1 hr following cocaine injection, coincident with the previously observed onset of increased conjugated diene absorption in microsomal lipids. During the previously observed period of peak conjugated diene absorption (2 to 4 hr), ultrastructural changes in centrilobular hepatocytes progressed. These included focal mitochondrial membrane disruption followed by more extensive mitochondrial swelling and disruption with increased swelling of rough endoplasmic reticulum. Changes in size, shape and concentration of histochemically labeled, morphometrically studied peroxisomes were also seen during this interval. Injury of centrilobular hepatocytes advanced to cell death in 6 to 8 hr. The time course and nature of these morphologic findings correlate with previously observed evidence of lipid peroxidation, supporting the hypothesis that this is the mechanism of cocaine hepatoxicity.

Effect of freezing and thawing on the microcirculation and capillary endothelium of the hamster cheek pouch

Golden Syrian hamster cheek pouches were prepared in vivo for observation of microvasculature under the light microscope. On an apparatus designed especially for this purpose, pouches were cooled from plus 10 °C to minus 30 °C at 1 °C/min, held at minus 30 °C for 1 min and warmed at 1 °C/min to plus 10 °C whence spontaneous rewarming to ambient was permitted. Pouches were observed under the light microscope throughout freezing and for 1 hr after thaw. At 0, 1, 5, 15, 30, and 60 min after thaw pouch tissue was taken for electron microscopy. After thaw hemostasis evolved in a biphasic pattern. In the early phase in which the degree of stasis reached its peak at about 20 min after thaw, cessation of circulation was caused by obstructive embolic platelet aggregates and a second uncharacterized factor, possibly humoral in nature. After 30 min, blood flow spontaneously resumed in at least one portion of each pouch. In 6 specimens, by 45–50 min, blood flow had returned to normal in about 90% of the microvasculature. After 50 min hemostasis began again and by 60 min blood flow had ceased in virtually all areas of the pouch, this time in the absence of vascular obstruction by platelet aggregates. Ultrastructural damage to endothelial cells occurring immediately at thaw and progressing through 1 hr thereafter included the rupture of cell membranes, early thinning, and later condensation of ground substance, decrease in the number and concentration of pinocytotic vesicles, swelling of rough endoplasmic reticulum, and the swelling and loss of cristal structure of mitochondria. This endothelial destruction can account for the permanent hemostasis of frostbite.