Hydration state of goats transported by road for 12 hours during the hot-dry conditions and the modulating role of ascorbic acid.

This study investigated the effects of 12 hr of road transportation during the hot-dry conditions and the modulating role of ascorbic acid (AA) on the hydration state of goats. Twenty goats who served as treatment goats received oral administration of 100 mg/kg body weight of AA, whereas another 20 control goats received sterile water; thereafter, the goats were loaded and transported. The study determined changes in skin thickness; albumin (Alb); total protein (TP); elimination of the gut content; fecal water; urine specific gravity (SG); and pH before, during, and after the transportation. The result obtained in the control goats showed significant (p < .05) increases in the values of TP, Alb, urine SG, and pH; elimination; and drinking behavior, whereas skin thickness decreased over transportation. In the treatment goats who were administered AA, the changes observed in the values were insignificant (p > .05). In conclusion, 12-hr road transportation of goats induced dehydration, which may affect the welfare and health status of the goats. The administration of AA ameliorated the risk of adverse effects of handling, loading, transportation, and hot-dry conditions on hydration state of goats.

The effect of hetastarch (670/0.75) on urine specific gravity and osmolality in the dog.

BACKGROUND: Urine specific gravity (USG) is used clinically to estimate urine osmolality (UOsm). Although USG has been shown to have a linear correlation with UOsm in dogs, the relationship is altered when there are significant numbers of high molecular weight (MW) molecules in the urine. HYPOTHESIS: USG would no longer predict UOsm in dogs given intravenous hetastarch (670/0.75)(HES). ANIMALS: Eight healthy employee-owned adult dogs. METHODS: Prospective, controlled experimental study. USG and UOsm were measured every 30 minutes from t=0 minutes to t=360 minutes. Dogs were administered 20 mL/kg of either NaCl 0.9% (control group, n=4) or HES (treatment group, n=8) IV over 1 hour starting at t=90 minutes. RESULTS: There was a decrease in UOsm in both groups starting at t=120 minutes and continuing for the study duration, and there was no significant difference in UOsm between treatment and control groups across all time points. There was an appropriate decrease in USG from t=120 minutes for the control group. In the treatment group, USG increased significantly at t=120 minutes (P= .0006), t=150 minutes (P= .0002), and t=180 minutes (P= .0044). The largest increase in USG occurred at t=150 minutes with a mean USG of 1.070 +/- 0.021 (range 1.038-1.104). CONCLUSIONS AND CLINICAL IMPORTANCE: Urine specific gravity should not be used to estimate urine solute concentration in dogs following the administration of 20 mL/kg of HES. In a clinical setting, the evaluation of USG following this dose of HES may lead to an overestimation of urine concentration.

Effect of magnesium, MK-801 and combination of magnesium and MK-801 on blood-brain barrier permeability and brain edema after experimental traumatic diffuse brain injury.

OBJECTIVE: Glutamate antagonists are very attractive drugs in laboratory works to protect neural tissue against ischemia. In this work, the effects of magnesium, MK-801 and combination of magnesium and MK-801 on blood-brain barrier (BBB) and brain edema after experimentally induced traumatic brain injury are evaluated. METHODS: A standard closed head injury was induced on the rats by a controlled impact device using a 450-g free falling mass from a height of 2 m onto a metallic disc fixed to the intact skull. One of the following was injected to animals intraperitoneally 30 minutes after injury: saline, magnesium, MK-801 and magnesium plus MK-801. To quantify the brain edema, the specific gravity of the brain tissue was determined. To demonstrate the alteration of the BBB permeability, Evans blue dye was used as a tracer. RESULTS: In all treatment groups, the specific gravity of brain tissue values was significantly higher compared with the control group. Evans blue dye content in the brain tissue was significantly reduced in all three treatment groups with respect to the control group. There was no significant difference of effect between the groups of magnesium alone and MK-801 alone when compared with each other and when compared with their combination. CONCLUSION: The present data demonstrate that treatment with magnesium, MK-801 and combination of magnesium and MK-801 can reduce formation of brain edema and can help restore BBB permeability after experimental diffuse brain injury.

Conivaptan bolus dosing for the correction of hyponatremia in the neurointensive care unit.

INTRODUCTION: Hyponatremia frequently complicates acute brain injury and may precipitate neurological worsening by promoting cerebral edema. An increase in brain water may be better managed through water excretion than with fluid restriction or hypertonic fluids. Vasopressin-receptor antagonists such as conivaptan, which promote free water excretion, may be ideal agents to treat this common and potentially serious disorder. METHODS: The efficacy of intermittent bolus doses of conivaptan to correct hyponatremia was examined in a consecutive series of patients treated in our neurointensive care unit. Patients were excluded if baseline sodium was over 135 mEq/l or if another conivaptan dose was given within 12 h. We assessed the proportion responding with a 4 or 6 mEq/l rise in sodium by 12 h, the change in sodium from baseline, and, in those not receiving another dose for at least 72 h, the long-term ability of a single dose to maintain sodium at least 4 mEq/l above baseline. We also recorded the effects of conivaptan on urine output and specific gravity, and noted any adverse events. RESULTS: A total of 25 doses given to 19 patients were included (out of 44 total doses administered in the study period). Sodium rose by 5.8 +/- 3.2 mEq/l within 12 h, with 71% rising by at least 4 mEq/l and 52% manifesting at least a 6 mEq/l increase. In those receiving only a single dose, 69% maintained at least a 4 mEq/l rise up to 72 h. Conivaptan also consistently led to increased urine output and a significant drop in urine specific gravity (i.e., aquaresis). No cases of phlebitis were observed despite administration of conivaptan through peripheral IVs. CONCLUSION: Intermittent dosing of conivaptan was effective in increasing free water excretion and correcting hyponatremia in neurologically ill patients. This supports its further evaluation for managing hyponatremia in this population.

Various irrigation fluids affect postoperative brain edema and cellular damage during experimental neurosurgery in rats.

BACKGROUND: This study was conducted to investigate how various irrigation fluids used during neurosurgical procedures affect the degree of postoperative brain edema and cellular damage during experimental neurosurgery in rats. METHODS: The cerebral cortex was exposed and incised crosswise with a surgical knife under irrigation with an artificial CSF, lactated Ringer's solution, or normal saline. Four hours after injury, irrigation was stopped and brain tissue samples were obtained from injured and uninjured sites. Specific gravity, cerebrovascular permeability, and TTC staining of the samples were evaluated. Incision and irrigation of the brain were not performed on the control group. RESULTS: At the injured site, specific gravities of the samples in the normal saline group and the lactated Ringer's solution group were significantly lower than the specific gravity in the artificial CSF group. The EB concentration was significantly higher in the lactated Ringer's solution group and relatively high in the normal saline group as compared with the artificial CSF group. TTC staining did not differ significantly between the artificial CSF group and the control group. It was significantly lower in the lactated Ringer's solution group and the normal saline group than in the control group and the artificial CSF group. CONCLUSIONS: As compared with normal saline and lactated Ringer's solution, artificial CSF reduced postoperative brain edema, cerebrovascular permeability, and cellular damage in sites injured by experimental neurosurgery in rats.

Effects of dietary protein intake on indexes of hydration.

This study aims to characterize the relationship between increased protein intake and hydration indexes. Five men participated in a 12-week, randomized, crossover, controlled diet intervention study. Subjects consumed eucaloric diets containing 3.6 (high protein), 1.8 (moderate protein), and 0.8 (low protein) g/kg/day of protein for 4 weeks each. Energy intakes were based on requirements established relative to resting energy expenditure and activity at baseline. Assessments included blood urea nitrogen, plasma osmolality, urine-specific gravity, and estimates of fluid balance. Repeated-measures analyses of variance and paired t tests were used to determine effects of treatment and time. Fluid intake and fluid balance were unaffected. Blood urea nitrogen was higher for high protein vs low protein and vs moderate protein, and urine-specific gravity was higher for high protein vs moderate protein. Baseline plasma osmolality was greater for high protein vs low protein and vs moderate protein. The effect of increasing dietary protein on fluid status was minimal.

Intraventricular albumin: an optional agent in experimental post-traumatic brain edema.

HYPOTHESIS: Human albumin may be effective in the treatment of posttraumatic brain edema due to its hyperoncotic features. Therefore, the aim of the experimental study presented in this paper has two points: the first is to evaluate the efficacy of intraventricular hyperoncotic human albumin on post-traumatic brain edema and the second is to try to show the appropriate posttraumatic time window for albumin administration. METHOD: Traumatic brain injury and subsequent edema was formed by a model of impact acceleration injury in rats. Human albumin was administered via intraventricular route by using a stereotactic head holder. All animals in each group were decapitated 24 hours after the procedure and the effect of albumin was evaluated by measurement of tissue specific gravity. RESULTS: Tissue specific gravity decreased in edematous tissue (trauma indicator), increased after albumin administration at the 12th (p < 0.001), and both at the 1st and 12th hour of the trauma (edema treatment; p < 0.001). On the other hand, albumin administered at the 12th, and at both the 1st and 12th hours in the rats without trauma has caused the formation of the brain edema. CONCLUSION: We conclude that human albumin is effective in cytotoxic, but not in vasogenic edema and exerts its best anti-edematous effect at the 12th hour of severe head trauma and this study may help future studies that will try to show the effects of albumin with different time modalities after severe head injury.

Studies of the toxicological potential of tripeptides (L-valyl-L-prolyl-L-proline and L-isoleucyl-L-prolyl-L-proline): IV. Assessment of the repeated-dose toxicological potential of synthesized L-valyl-L-prolyl-L-proline in male and female rats and dogs.

The objective of these repeated-dose, 8-week studies was to assess the toxicological potential of a synthetic tripeptide, L-valyl-L-prolyl-L-proline (VPP), when administered to Charles River rats and Beagle dogs. Groups of 20 male and 20 female rats were fed powdered diets containing sufficient VPP to afford daily doses of 0, 2, 8, or 16 mg/kg body weight (BW)/day. Groups of five male and five female dogs were administered 0, 2, 8, or 16 mg/kg BW/day in hard gelatin capsules. Antemortem evaluative parameters for both species included grossly observable clinical signs, body weight and food consumption, clinical pathology (hematology, clinical chemistry, urinalysis), and ophthalmological examinations. Dogs also received electrocardiographic examinations. Postmortem evaluations in both species included complete necropsy, determination of major organ weights, and histopathological examination of specimens from approximately 50 organs and tissues. All rats and dogs survived to the scheduled termination of the studies and neither species exhibited evidence of VPP effects on appetite or body weight gain/maintenance. Ophthalmic examinations revealed occasional lens clouding in rats, but this occurred in all groups and was not attributable to VPP. Some clinical pathology parameters in both species were occasionally altered, but there was no evidence that this was dose-related. Electrocardiographic examinations in dogs revealed no VPP-associated changes. Mid- and high-dose male rats (but not females) had slightly reduced mean pituitary and kidney weight parameters, whereas mid- and high-dose females had slightly increased mean uterus:body weight ratios. There were no microscopic correlates for these minor changes. Ten percent to 20% of all female rats (but not males) exhibited corticomedullary mineralization of the kidney and gliosis of the optic nerve, and 10% to 20% of males (but not females) had thymic hemorrhage. Postmortem evaluations of dogs revealed no VPP-related effects on organ weights or either macro- or microscopic appearances of organs. The results of these studies provided no evidence of either local or systemic toxicity. Similarly, there was no evidence of neurotoxicity that might have been detected by the appearance of physical or behavioral changes during gross observations of animals. Although these results do not identify target organs for VPP toxicity, the no-observable-effect level and maximally tolerated dose are both greater than 16 mg/kg/day when administered to male and female rats and dogs for 8 consecutive weeks. Based upon food enhancement levels of VPP currently being evaluated, the resultant margin of safety (160) is substantial.

Pharmacokinetics and pharmacodynamics of furosemide after oral administration to horses.

Furosemide is the most common diuretic drug used in horses. Furosemide is routinely administered as IV or IM bolus doses 3-4 times a day. Administration PO is often suggested as an alternative, even though documentation of absorption and efficacy in horses is lacking. This study was carried out in a randomized, crossover design and compared 8-hour urine volume among control horses that received placebo, horses that received furosemide at 1 mg/kg PO, and horses that received furosemide at 1 mg/kg IV. Blood samples for analysis of plasma furosemide concentrations, PCV, and total solids were obtained at specific time points from treated horses. Furosemide concentrations were determined by reversed-phase high-performance liquid chromatography with fluorescent detection. Systemic availability of furosemide PO was poor, erratic, and variable among horses. Median systemic bioavailability was 5.4% (25th percentile, 75th percentile: 3.5, 9.6). Horses that received furosemide IV produced 7.4 L (7.1, 7.7) of urine over the 8-hour period. The maximum plasma concentration of 0.03 microg/mL after administration PO was not sufficient to increase urine volume compared with control horses (1.2 L [1.0, 1.4] PO versus 1.2 L [1.0, 1.4] control). There was a mild decrease in urine specific gravity within 1-2 hours after administration of furosemide PO, and urine specific gravity was significantly lower in horses treated with furosemide PO compared with control horses at the 2-hour time point. Systemic availability of furosemide PO was poor and variable. Furosemide at 1 mg/kg PO did not induce diuresis in horses.

[Cell shrinkage during apoptosis is not obligatory. Apoptosis of U937 cells induced by staurosporine and etoposide]. / Degidratsionnoe sokrashchenie ob"ema kletok pri apoptoze--fakul'tativnyi priznak. Apoptoz kletok U937, vyzvannyi staurosporinom i étopozidom.

A study was made of apoptotic cell shrinkage, which is generally believed to be a hallmark of apoptosis. The two conventional models of apoptosis were used for examination of changes in cell water balance--one is apoptosis caused in human lymphoma cell line U937 by staurosporine, and the other by etoposide. Intracellular water was determined by measuring buoyant density of cells in continuous Percoll gradient. Apoptosis was recognized by microscopy and flow cytometry. Apoptosis caused by staurosporine (1 microM, 4 h) was found to be associated with a decrease in cell water content by almost 24%. In contrast, no decrease in cell water content was observed in U937 cells incubated with etoposide (50 microM, 4 h), in spite of the number of features suggesting the presence of apoptosis, such as the appearance of apoptotic bodies, chromatin condensation and fragmentation and disappearance of S-phase cells in DNA histogram. It is concluded that definition of apoptosis as "shrinkage-necrosis" (Kerr, 1971) needs correcting: the distinction of apoptotic cells involves the absence of swelling, rather than cell shrinkage.

Effect of alfalfa meal in diets of laying quails on performance, egg quality and some serum parameters.

This study was conducted to investigate the effects of increasing levels of alfalfa meal in the diet of laying quails on egg production, feed consumption, feed efficiency, egg quality, egg yolk cholesterol and selected serum parameters. In this study, 192, 10-week old quails (Coturnix coturnix japonica) were evenly distributed to four groups with four replicates of 12 quails each. The control group was fed a basal diet containing 0% alfalfa meal and the remaining groups received 3, 6 or 9% alfalfa meal for 12 weeks. Live weight, feed consumption, and egg production were recorded and feed efficiency were calculated. Eggs were examined for interior and exterior quality and egg yolk cholesterol content. At the end of the experiment, blood samples were collected and sera were analysed for serum Ca, inorganic P (P(i)), Mg, triglycerides and total cholesterol. Any level of alfalfa meal had no effect on live weight, egg production, feed consumption, feed efficiency, egg weight, and egg yolk index. Six percent and 9% alfalfa meal increased specific gravity of whole egg and eggshell thickness as well as serum P(i) levels. Nine percent alfalfa meal reduced serum triglycerides, total cholesterol levels and egg yolk cholesterol content. The results of this experiment indicated that addition of 9% alfalfa meal into the laying quail diet may improve eggshell quality and reduced serum triglycerides and serum and egg yolk cholesterol without any adverse effect on performance.

Furosemide continuous rate infusion in the horse: evaluation of enhanced efficacy and reduced side effects.

Continuous rate infusion (CRI) of furosemide in humans is considered superior to intermittent administration (IA). This study examined whether furosemide CRI, compared with IA, would increase diuretic efficacy with decreased fluid and electrolyte fluctuations and activation of the renin-angiotensin-aldosterone system (RAAS) in the horse. Five mares were used in a crossover-design study. During a 24-hour period, each horse received a total of 3 mg/kg furosemide by either CRI (0.12 mg/kg/h preceded by a loading dose of 0.12 mg/kg IV) or IA (1 mg/kg IV q8h). There was not a statistically significant difference in urine volume over 24 hours between methods; however, urine volume was significantly greater after CRI compared with IA during the first 8 hours ([median 25th percentile, 75th percentile]: 9.6 L [8.9, 14.4] for CRI versus 5.9 L [5.3, 6.0] for IA). CRI produced a more uniform urine flow, decreased fluctuations in plasma volume, and suppressed renal concentrating ability throughout the infusion period. Potassium, Ca, and Cl excretion was greater during CRI than IA (1,133 mmol [1.110, 1,229] versus 764 mmol [709, 904], 102.7 mmol [96.0, 117.2] versus 73.3 mmol [65.0, 73.5], and 1,776 mmol [1,657, 2.378] versus 1,596 mmol [1,457, 1,767], respectively). Elimination half-lives of furosemide were 1.35 and 0.47 hours for CRI and IA, respectively. The area under the excretion rate curve was 1,285.7 and 184.2 mL x mg/mL for CRI and IA, respectively. Furosemide CRI (0.12 mg/kg/h) for 8 hours, preceded by a loading dose (0.12 mg/kg), is recommended when profound diuresis is needed acutely in horses.

Body composition and vascular effects of growth hormone administration in old female rats.

Aging is associated with alterations in cardiovascular system and changes in body composition. The aim of this study was to investigate the effect of GH on body composition, vascular function and structure in old female rats. Old (20 months) and adult (4 months) female Wistar rats were used. One group of old animals was treated with GH (2 mg/kg/day) for four weeks. Periuterine fat weight, specific gravity index (SGI), dose response to Acetylcholine, Isoprenaline, Phenylephrine and Acetylcholine in the presence of L-NAME and vascular morphology in aortic rings, were studied. Old rats showed increased fat weight and decreased SGI (p<0.05) as compared to adult animals. GH reduced fat weight (p<0.05) and tended to increase SGI (NS). Old rats showed impaired vasodilatation to Acetylcholine and Isoprenaline (p<0.05), and GH improved these responses (p<0.05). Contraction response to Phenylephrine was higher in old than in adults rats (p<0.05), but GH did not show any effect. Contraction induced by Acetylcholine+L-NAME was higher in old rats than in adults, and GH tended to reduce this response, although not significantly. Aortic media area was increased in old rats, and GH reduced this parameter (p<0.05). In conclusion, GH shows beneficial effects on body composition, vascular function and morphology in old female rats.

Detection, quantification, and pharmacokinetics of furosemide and its effects on urinary specific gravity following IV administration to horses.

Furosemide is a potent loop diuretic used for the prevention of exercise-induced pulmonary hemorrhage in horses. This drug may interfere with the detection of other substances by reducing urinary concentrations, so its use is strictly regulated. The regulation of furosemide in many racing jurisdictions is based on paired limits of urinary SG (<1.010) and serum furosemide concentrations (>100 ng/ml). To validate this regulatory mechanism, a liquid chromatography/mass spectrometry/mass spectrometry method employing a solid-phase extraction procedure and furosemide-d5 as an internal standard was developed. The method was used to determine the pharmacokinetic parameters of furosemide in equine serum samples and its effects on urinary SG after IV administration (250 mg) to 10 horses. Pharmacokinetic analysis showed that serum concentrations of furosemide were well described by a two-compartmental open model. Based on results in this study, it is very unlikely for horses to have serum furosemide concentrations greater than 100 ng/ml or urine SG less than 1.010 at 4 hours after administration (250 mg IV). However, it should be remembered that urine SG is a highly variable measurement in horses, and even without furosemide administration, some horses might naturally have urine SG values less than 1.010.

Effect of furosemide on urine specific gravity and osmolality in thoroughbred racehorses.

Postrace urine samples from thoroughbred horses were examined to compare osmolality and specific gravity between horses treated with furosemide and those not treated. Samples were assigned to groups in relation to reported medication (furosemide) status, race finish position, and distance of race. Urine osmolality was significantly (P <.05) lower in samples from horses treated with furosemide when compared with untreated horses. Specific gravity determinations are less precise at measuring urine osmolality at lower levels (1.01 g/ml or less). The measurement of osmolality is a superior method for determining the urine solute concentration and facilitating the regulation of furosemide.

Effects of antioxidant, OPC-14117, on secondary cellular damage and behavioral deficits following cortical contusion in the rat.

In the present study, we examined the effects of OPC-14117, a superoxide radical scavenger, on the secondary cellular damage and cognitive dysfunction occurring in a rat model of cerebral contusion induced by a controlled cortical impact (CCI). Histological examinations revealed that the contusion necrosis volume reached 13.6+/-5.3 mm(3) in non-treated animals and declined to 1.9+/-0.6 mm(3) in OPC-14117-treated animals (P<0.01). The cell number of the CA3 region was 120.0+/-12.4 cells/mm in the normal controls, 73.6+/-9.9 cells/mm in the non-treated animals, and 111.2+/-10.2 cells/mm in the OPC-14117-treated animals, indicating that CCI-induced selective neuronal cell death in the CA3 region was attenuated by the OPC-14117 administration (P<0.01). The tissue osmolality, as determined with a vapor pressure osmometer, was 314.5+/-15.4 mmol/kg in the normal brain and increased to 426.0+/-20.1 mmol/kg at 12 h following CCI. The increase in tissue osmolality was significantly attenuated by OPC-14117 administration (P<0.01). The OPC-14117 administration also attenuated the CCI-induced cognitive deficits. The OPC-14117-treated animals showed a tendency to improve on the Morris water maze performance test. The impairment of the habituation of exploratory activity elicited by CCI was significantly attenuated by OPC-14117 administration (P<0.05). In conclusion, OPC-14117 may have a potential for decreasing secondary cellular damage due to traumatic brain injury since it is as efficacious as any other compound tested in this model.

Estimation of the probability for exceeding thresholds of urine specific gravity and plasma concentration of furosemide at various intervals after intravenous administration of furosemide in horses.

OBJECTIVE: To estimate the probability of concurrently exceeding thresholds for plasma concentration of furosemide and urine specific gravity after IV administration of furosemide in horses. ANIMALS: 12 mature healthy Thoroughbred (n = 6) or Quarter Horse (6) mares. PROCEDURE: Venous blood was collected from each horse prior to and 0.25, 0.5, 0.75, 1, 2, 3, 4, 4.5, 5, and 6 hours after IV administration of 250 mg (first experiment) or 500 mg (second experiment) of furosemide. Urine was collected hourly between 1 and 6 hours after administration of furosemide at both doses. Concentrations of furosemide were determined by use of an ELISA. Concentration of furosemide and urine specific gravity was modeled as a function of time, accounting for inter- and intrahorse variabilities. On the basis of pharmacokinetic and specific gravity data, the probability of exceeding a concentration of 100 ng of furosemide/ml as a function of time was determined, using a semiparametric smooth functional averaging method. A bootstrap approach was used to assess the inherent variation in this estimated probability. RESULTS: The estimated probability of exceeding the threshold of 100 ng of furosemide/ml and urine specific gravity < 1.012 was approximately 0% between 4.0 and 5.5 hours after IV administration of 250 mg of furosemide/horse, and ranged from 0 to 1% between 4 and 5.5 hours after IV administration of 500 mg of furosemide/horse. The probability of a horse being falsely identified as in violation of regulatory concentrations was inversely associated with time. CONCLUSIONS AND CLINICAL RELEVANCE: Coupling plasma furosemide concentration with urine specific gravity testing will greatly reduce the chance that some horses are misclassified as being in violation of regulatory concentrations.

Nephrogenic diabetes insipidus in a dog with intestinal leiomyosarcoma.

Nephrogenic diabetes insipidus was diagnosed in a dog with an intestinal leiomyosarcoma. The diagnosis of nephrogenic diabetes insipidus was made on the basis of results of serum biochemical tests, urinalyses, and a water-deprivation test, along with a lack of response to exogenous administration of vasopressin following the water-deprivation test. The temporal association between resection of the intestinal mass and resolution of clinical signs of diabetes insipidus (i.e., polyuria and polydipsia) and between recurrence of clinical signs and detection of metastatic disease suggests that there may have been a causal relationship, and nephrogenic diabetes insipidus may have developed as a paraneoplastic syndrome in this dog.

N omega-nitro-L-arginine attenuates early ischemic neuronal damage of prolonged focal cerebral ischemia and recirculation in rats.

The present study aimed to examine the effects of N omega-nitro-L-arginine (LNA) on the early ischemic neuronal damage (EIND). All the experiments were carried out under general anesthesia, maintaining the blood gases and the body temperature within the physiological ranges. The local CBF, the topographically corresponding cortical specific gravity, and the volume of EIND were determined in each rat, which was subjected to prolonged or temporary occlusion of middle cerebral artery (MCA) using our original miniclip. Significant cortical edema developed only in the brain area where the local CBF value was below 200 ml 100 g-1 min-1. The prolonged MCA occlusion for 1, 2, and 4 h induced a time-dependent increase in the severity of cortical edema and the volume of EIND. Removal of the clip invariably induced recirculation. Compared to that induced by 4 h prolonged ischemia, the brain damage was improved by 1 h MCA occlusion followed by 3 h recirculation, whereas it was significantly worsened by 2 h ischemia followed by 2 h recirculation. While LNA [1 mg, i.p., given two times during the experiment] only partially inhibited the activity of brain nitric oxide synthase, it remarkably ameliorated EIND of both prolonged ischemia and recirculation in this model. The above findings indicate the pathogenic role of nitric oxide in prolonged ischemia as well as recirculation.