Cholinergic drug interactions and heat tolerance.

Acute, subchronic and chronic exposures to cholinergic compounds may result in differing effects. The efficacy of pyridostigmine bromide (PY) prophylaxis against organophosphorus poisoning depends on post exposure atropine (AT) administration. AT induces a dose-dependent increase in rate of rise of core temperature in heat exposed humans and rats. To determine whether AT's anticholinergic potency is altered following PY administration, we examined AT's effects following acute or subchronic (2 weeks) PY administration in the sedentary heat-stressed rat. Four groups of rats were acutely (a, i.v.) treated with saline (SAL) or PY (100 ug/kg) followed by SAL or AT (200 ug/kg), and 4 groups were subchronically (c, osmotic pump) treated with SAL or PY (20 ug/hr) followed by SAL or AT (200 ug/kg). Fifteen minutes following the final injection, rats were subjected to an ambient temperature of 41.5 degrees C until a core temperature of 42.6 degrees C was attained. Heat tolerance times were significantly improved for cPY+SAL over aPY+SAL (241 +/- 9 vs 187 +/- 16 min, mean +/- SE) and for cPY+AT over aPY+AT (76 +/- 9 vs 57 +/- 2 min). The improvement in thermoregulation resulted from increased salivary water for evaporative cooling indicated by % weight loss (corrected for fecal loss) during heat stress: cPY+SAL over aPY+SAL (8.4 +/- 0.3 vs 6.6 +/- 0.5%). This increased heat tolerance resulting from subchronic anticholinesterase administration resembles changes seen with heat acclimation.

Cooling to heat of fusion (HOF), followed by rapid rewarming, does not reduce the integrity of microvascular corrosion casts.

This study utilized microvascular corrosion casting techniques to evaluate changes in the microvascular patency of rat hindpaws cooled to four different subzero temperatures. Left hindpaws of anesthetized rats in group 1 were cooled to -5 degrees C, in group 2 to -15 degrees C, in group 3 to heat of fusion (HOF), and in group 4 to HOF and then to -15 degrees C. Although freezing did not take place in the hindpaws of groups 1 and 2, initiation of freezing in the tissues, as indicated by HOF, did occur in groups 3 and 4. Cooled hindpaws were rapidly rewarmed. Right hindpaws served as controls. Microvascular corrosion casts were made from the left and right hindpaws of all animals. There was no significant difference when the mean cast weights of cooled hindpaws from groups 1, 2, and 3 were compared to the mean cast weights of their respective control hindpaws. In group 4, there was a significant difference (P less than 0.05) when the mean cast weight of the cooled hindpaws (47.69 +/- 9.05, mg +/- SEM) was compared to that of the control hindpaws (80.63 +/- 12.23). Since, in this acute experiment, a loss of vascular integrity occurred when the hindpaws in group 4 were cooled to -15 degrees C after reaching HOF, the initiation of freezing alone was not sufficient to reduce mean cast weight.

Chronic vs acute carbamate administration in exercising rats.

Physostigmine (PH), alone, and pyridostigmine (PY), in combination with atropine and 2-PAM, have been shown to protect animals against organophosphate poisoning. While acute administration of either of these carbamates increased heating rates and decreased endurance of exercising rats, chronically administered PY did not induce these decrements, and we hypothesized that chronic administration of PH could also result in similar attenuation of these effects. Thus, PH was administered acutely (iv) or chronically (osmotic mini-pump) in the following 4 groups (510-530g, male, N = 10/group): C (control, saline iv), AC-200 (acute, 200 ug/kg, 58% whole blood cholinesterase (ChE) inhibition), CH-7 (chronic, 125 ug/hr, 7 days, 60% inhib.), and CH-14 (chronic, 125 ug/hr, 14 days, 56% inhib.). Rats were run (11 m/min, 26 degrees C) to exhaustion. The run times and heating rates (% of control) were: AC-200 - 47, 213%; CH-7 - 60, 157%; CH-14 - 92, 109%. Additionally, ultrastructural changes noted in diaphragms of acutely treated animals were less evident in chronically treated animals. Thus, the decremental effects of acute PH administration on endurance, thermoregulation, and ultrastructure were attenuated with chronic administration at similar levels of ChE inhibition.

An evaluation of the ability of the peripheral vasodilator buflomedil to improve vascular patency after acute frostbite.

The extent of microvascular damage from frostbite can be accurately demonstrated by vascular microcorrosion casting techniques (P. S. Daum, W. D. Bowers, Jr., J. Tejada, and M. P. Hamlet, Cryobiology 24, 65-73, 1987). In the present investigation, the peripheral vasodilator buflomedil was evaluated for its ability to ameliorate microcirculatory damage from acute experimentally induced freeze injury. This drug has been reported to decrease tissue loss in human frostbite patients when given intravenously during thawing (J. Foray, P. E. Baisse, J. P. Mont, and Cl. Cahen, Sem. Hop. Paris 56, 490-497, 1980). In seven groups of anesthetized rats, left hindpaws were cooled to heat of fusion; cooling continued until the temperature in the footpads fell to -15 degrees C. Prior to cooling, group 1 received a tail vein injection of 1 ml saline/kg, while group 2 received 10 mg buflomedil/kg. Immediately following cooling, group 3 received an injection of 10 mg buflomedil/kg. Hindpaws were rapidly rewarmed in a 40 degree C bath. During rewarming, left hindpaws from group 4 were immersed in deionized water, from group 5 in 24 mg buflomedil in deionized water, from group 6 in 30% dimethyl sulfoxide (Me2SO), and from group 7 in 24 mg buflomedil in 30% Me2SO. Right hindpaws served as controls. Vascular microcorrosion casts were made from left and right hindpaws of all groups. There was no significant difference in mean cast weights when frozen hindpaws of the seven groups were compared, although treatment with buflomedil increased the mean cast weight of control hindpaws from groups 3 and 7. It therefore appears that, in this acute model for frostbite, buflomedil does not improve vascular patency.

Vascular casts demonstrate microcirculatory insufficiency in acute frostbite.

The use of vascular microcorrosion casts (vascular replicas) has made it possible to demonstrate the degree of damage to the microcirculation in experimentally induced frostbite. This approach provides a direct method for demonstrating vascular patency. Four groups of animals were used in this investigation. The left hind limbs of anesthetized rats were cooled to -10 degrees C in groups one and three and to -20 degrees C in groups two and four, as measured by needle thermocouples placed under the gastrocnemius muscles. Thermocouples were also placed in the left hind footpads of groups three and four. The sheathed limbs were cooled in an alcohol bath at approximately 1.1 degree C per minute. All limbs exposed to the cold bath were rewarmed to 37 degrees C in a 40 degree C water bath. The right hind limbs served as uninjured controls. The footpad temperatures recorded in groups three and four were used in conjunction with the temperatures recorded under the gastrocnemius muscles to characterize the footpad temperatures in groups one and two. Vascular microcorrosion casts were made from the left and right hind paws of groups one and two using Batson's modified methyl methacrylate. Scanning electron microscopic examination of the casts demonstrated dramatic differences between the vascular integrity of control paws and that of frozen paws. Exposure to the cold temperatures destroyed most of the microcirculation. In addition, the weights of the casts from the control paws were significantly different from the weights of the casts from the frozen paws. It was concluded that this model for evaluating frostbite injury accurately demonstrates the extent of microvascular damage and has significant potential as a method for evaluating therapeutic drug regimens.

Routes of viral spread in the von Szily model of herpes simplex virus retinopathy.

Intraocular inoculation of herpes simplex virus type 1 (HSV-1) in one eye of rabbits results in encephalitis and contralateral necrotizing viral retinopathy. The effects of viral inoculation site and optic nerve (ON) transection on the spread of virus to the brain and contralateral eye in this model were investigated. A surgical technique was developed for transection of the retrobulbar optic nerve posterior to the entrance of the central retinal vessels. HSV-1 was inoculated into the AC or vitreous of one eye in normal rabbits and in rabbits with one ON transected, either ipsilateral or contralateral to the side of inoculation. Animals were followed clinically for signs of disease. Encephalitis and contralateral retinopathy (CR) occurred following both AC and vitreous inoculation of virus, although CR developed later in AC-inoculated rabbits. Ipsilateral retinopathy (IR) developed in 83% of vitreous-inoculated rabbits, but in only 5% of AC-inoculated animals. IR developed 8 days after the onset of CR in the AC-inoculated group. ON transection on the side of virus inoculation prevented development of CR only in vitreous-inoculated rabbits. ON transection on the side opposite virus inoculation prevented CR regardless of the site of inoculation. These findings suggest that HSV-1 can leave the inoculated eye by multiple routes depending on the site of virus inoculation, but that virus reaches the retina of the contralateral eye via the optic nerve.

Effect of low-potassium diet on rat exercise hyperthermia and heatstroke mortality.

A total of 182 male Sprague-Dawley rats weighing 250-300 g were fed either a control (n = 122) diet for 32 days. The diets contained either 125 or 8 meq potassium/kg, respectively. Rats fed the low-K diet gained weight at only one-third the rate of controls (1.7 vs. 5.2 g/day), and their skeletal muscle and plasma potassium levels were reduced by 28 and 47%, respectively. When run to exhaustion at either 15 or 20 degrees C, low K+-fed rats accomplished less than one-half of the work done by the controls (26 vs. 53 kg. m) but exhibited a markedly greater rate of heat gain per kilogram-meter of work than controls (0.12 vs. 0.05 degrees C)ambient temperature of 20 degrees C, the rats of the low-K+ group despite large differences in body weight (-25%), run time temperature and twice (33 vs 17%) the mortality rate of the controls. Postexercise increases in circulating potassium (less than 90%) of heat-injured rats raised the plasma levels of low K+-fed rats to normal (5.9 +/- 2.2 meq/l). These results appear to characterize the existence of an insidious and, therefore, undocumented form of fatal exertion-induced heat illness.

Diagnostic significance of selected serum enzymes in a rat heatstroke model.

A total of 171 untrained, unacclimatized, and unanesthetized rats were either exercised to exhaustion at one of four ambient temperatures (5, 20, 26, or 30 degrees C), or were restrained and heated at an ambient temperature of 41.5 degrees C until their core temperatures reached a preselected end point between 41.0 and 43.3 degrees C. The serum levels of creatine phosphokinase (CRK) and two transaminases (SGOT and SGPT) were determined at 30 min, 24, 48, 72, and 96 h posttreatment. Peak enzyme activity for CPK was noted primarily at the 30-min sampling period and at 24 h for the transaminases. The data indicated that under these conditions a) the transaminase SGOT was elevated in the serum as a consequence of the extent and duration of prior hyperthermia, b) the transaminase SGOT was released in moderate amounts after exhaustive exercise but reached its greatest activity levels following hyperthermia, and c) the activity of CPK was increased by the duration of exhaustive exercise and was less sensitive than either transaminase to prior hyperthermia. As a result, each of the three experimental conditions: a) exercise without hyperthermia, b) exercise with hyperthermia, and c) sedentary hyperthermia, produced a unique pattern of serum enzyme activity that would appear useful in diagnosing a variety of heat- and/or work-induced disorders.

Role of physical effort in the etiology of rat heatstroke injury and mortality.

A total of 171 untrained, unacclimatized, and unanesthetized rats were used to evaluate the effects of sedentary and work-induced hyperthermia on the incidence of mortality and cellular injury, 24 h postexposure. Cellular injury was defined as serum transaminase activity (SGPT and SGOT) exceeding 1,000 IU/l (heatstroke levels). Both the percent mortality and the percentage of 24-h survivors with transaminase levels above 1,000 IU/l were plotted against maximum core temperatures. Exertion-induced hyperthermia produced a significantly higher incidence of cellular injury and heatstroke death at lower core temperatures than hyperthermia alone. With hyperthermia only, the SGPT and SGOT dose-response curves were identical. When work was combined with hyperthermia, there was a greater incidence of elevated SGOT at lower core temperatures. These curves bore a striking resemblance to curves reflecting heat- and/or work-induced mortality in humans. The results suggest a direct role of physical effort in causing heatstroke injury and mortality.

Alterations of rat liver subsequent to heat overload.

Since pathological changes in the liver are among the consistent findings in humans subsequent to heatstroke, specimens were taken from the liver in rats during a study to assess the rat as a model for human heatstroke. Tissues from four groups of rats were processed for light and electron microscopy. The groups consisted of control rats, rats run to exhaustion at 5 C, rats exhausted at 26 C, and rats restrained at 41.5 C until their rectal temperatures reached 42.3 C. Exhaustive exercise at 5 C produced neither fatalities nor pathological changes in the livers. Exhaustive exercise at 26 C and restraint at 41.5 C were fatal for most rats. Histological and/or ultrastructural changes, which included centrilobular necrosis, vacuolization and diminution of hepatocellular microvilli, and loss of sinusoidal endothelium, were observed in livers from rats that were run to exhaustion at 26 C and from those rats restrained at 41.5 C. This work supports the validity of the rat model, since human heatstroke results in similar hepatic changes.

Rat model of acute heatstroke mortality.

A total of 252 untrained, unacclimatized, and unanesthetized laboratory rats weighing between 485 and 545 g were fasted and either run to exhaustion at 5, 20, 23, or 26 degrees C or were restrained and heated at an ambient temperature of 41.5 degrees C. The incidence of mortality associated with a wide range of work-induced hyperthermias was compared to the lethality of equivalent heat loads in the absence of physical effort. The severity of hyperthermia was calculated in degree-minutes above a base-line core temperature of 40.4 degrees C. The LD25's of run-exhausted versus restrained-heated rats were 16.8 and 30.1 deg-min, respectively. Survivors had a faster cooling rate than fatalities, but run-exhausted survivors had a slower cooling rate than restrained-heating survivors. Results indicate that 1) both the incidence of mortality and the survival time can be predicted from the severity of core heating, 2) work-related factors contribute to an increased rate of heatstroke death at low thermal loads, and 3) retrospectively, both heat-sensitive and heat-resistant groups were identified.

The laboratory rat as a model for hyperthermic syndromes in humans.

To assess the lethal effects of work-induced hyperthermia on exercising animals, untrained rats were run to exhaustion at 5 and 20-26 degrees C or restrained at 41.5 degrees C. An exercise-induced core temperature of 40.4 degrees C represented a base line above which mortalities occurred. With increasing core temperature at exhaustion (between 40.4 and 43 degrees C), mortality increased within 24 h. A dose-respones curve with an LD50 equivalent to a core temperature of 41.5 +/- 0.1 degrees C was calculated. Although differences in body weight loss, core temperature at exhaustion, and cooling rate will clearly distinguish between survivors and fatalities, the severity of heat injury as inferred from survival times is best measured by the time versus intensity of hyperthermia in degree-minutes.