Methods to Produce Brain Hyperthermia

Hari Shanker Sharma1

1 Institute of Surgical Sciences University Hospital, Uppsala University, Uppsala
Publication Name:  Current Protocols in Toxicology
Unit Number:  Unit 11.14
DOI:  10.1002/0471140856.tx1114s23
Online Posting Date:  March, 2005
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Abstract

With the increase in global warming, the problems of hyperthermia have recently attracted world‐wide medical attention. Deaths due to heat‐related illnesses that have occurred in many human populations in recent years are now recognized as a great social and medical problem. Interestingly, the detailed mechanisms of hyperthermia and probable therapeutic measures have still not been worked out. Thus, good experimental models to simulate hyperthermia under clinical conditions are needed to expand our knowledge in the field and to develop suitable therapeutic strategies in the future. This unit describes an animal model to induce hyperthermia that is comparable to the clinical situation. The model will be useful for studying the effects of heat‐related illnesses on various organs and systems. Because hyperthermia is associated with brain dysfunction, methods to assess some crucial parameters of brain injury, such as breakdown of the blood‐brain barrier and brain edema formation, are also described.

Keywords: hyperthermia; brain dysfunction; cell injury; blood‐brain barrier; brain edema; rectal temperature; anesthetics; heat stress; heat stroke

     
 
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Table of Contents

  • Basic Protocol 1: Producing Hyperthermia in the Unanesthetized Rat
  • Alternate Protocol 1: Hyperthermia Induced by an Infrared Heat Lamp
  • Alternate Protocol 2: Hyperthermia Induced in Heat Chambers using Anesthetized Animals
  • Support Protocol 1: Postmortem Evaluation of Heat Stress: Microhemorrhages in the Stomach
  • Assessing Changes in Brain Function After Heat Exposure
  • Support Protocol 2: Measuring Blood‐Brain Barrier Permeability to Evans Blue
  • Support Protocol 3: Measurement of Brain Water Content
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
  • Tables
     
 
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Materials

Basic Protocol 1: Producing Hyperthermia in the Unanesthetized Rat

  Materials
  • Rats or mice (age and sex controlled)
  • Paraffin oil or glycerine
  • 70% (v/v) ethanol
  • Thermistor probe (see ) suitable for rats or mice
  • Waterproof marker
  • Plastic cage, medium size
  • Thermometers, digital or ordinary mercury, with ±0.1°C accuracy
  • Biological oxygen demand (BOD) incubator (e.g., BS Pyromatic India, Asco, Raj Scientific Industries) or comparable heat chamber, preheated to desired temperature (e.g., 38°C)

Alternate Protocol 1: Hyperthermia Induced by an Infrared Heat Lamp

  • Anesthetic (see and see )
  • 75‐, 150‐, or 200‐W infrared lamp

Alternate Protocol 2: Hyperthermia Induced in Heat Chambers using Anesthetized Animals

  • Anesthetic (see and see )

Support Protocol 1: Postmortem Evaluation of Heat Stress: Microhemorrhages in the Stomach

  Materials
  • Rats or mice, control and heat‐exposed
  • Equithesin solution (see recipe)
  • 2% (w/v) Evans blue solution (see recipe)
  • Physiological (0.9% w/v NaCl) saline, room temperature and 4°C
  • 4% (w/v) paraformaldehyde fixative (see recipe), optional
  • 1‐ml glass or plastic syringes, sterile
  • 26‐ to 28‐G needle (o.d., 0.3 to 0.4 mm)
  • Surgical instruments:
    • Scalpel
    • Forceps
    • Fine scissors
  • Magnifying glass or stereomicroscope
  • Cotton wool, sterile
  • Intravenous perfusion setup, including infusion needle
  • 21‐G butterfly cannula (o.d., 0.8 mm)
  • 2‐ or 3‐way connector, optional
  • Additional reagents and equipment for sectioning rat brain, optional

Support Protocol 2: Measuring Blood‐Brain Barrier Permeability to Evans Blue

  Materials
  • Dissected rat brains after treatment with Evans blue (see protocol 5), from both control and heat‐stressed animals
  • Laboratory oven, 90°C
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Figures

  •   FigureFigure 11.14.1 Changes in (AC) body temperature, (D) pain perception, (E) body weight, and (F) salivation in control and heat‐stressed rats. (A) Handling alone alters the rectal temperature for 1 to 4 days; the rats then adapt to handling stress from day 5 onwards. A minimum of 7 days handling is necessary to avoid stress in animals. (B) Subjection of rats to heat stress at 38°C in a biological oxygen demand (BOD) incubator results in graded hyperthermia that is most severe after 4 hr. (C) The magnitude of hyperthermia is considerably less when rats are exposed to heat at 36°C. (D) Measurement of tail flick response in heat‐exposed rats at 38°C did not show an analgesic or hyperalgesic response. This indicates that heat exposure did not influence normal pain pathways. (E) Changes in body weight during heat exposure of rats at 38°C. Less intake of water and evaporative heat loss through salivation appear to be the important factors in reducing the weight of rats exposed to heat stress. A significant reduction in body weight is seen after 4 hr of heat exposure. (F) Spread of saliva over snout of rats exposed to heat at 38°C. The area of spread (in millimeters) is maximum following 4 hr heat exposure in rats. This suggests that rats subjected to 4 hr heat stress did not develop heat stroke, which stops the production of saliva (see ). * P < 0.05, ** P < 0.01; ANOVA followed by Dunnet's test for multiple group comparison with one control group (A‐E) or with the 30‐min group (F). Values are mean ± SD of six to eight rats in each group.
  •   FigureFigure 11.14.2 Representation of Evans blue albumin extravasation on the (A,C) dorsal and (B,D) ventral surfaces of the rat brain after 4 hr heat stress. Scale bar in B is 4 mm. Different regions of the cerebral cortex are divided using hypothetical solid black lines; extravasation is indicated by the shaded regions. Reprinted from The Blood‐Spinal Cord and Brain Barriers in Health and Disease (H.S. Sharma and J. Westman, eds.), H.S. Sharma (), Influence of serotonin on the blood‐brain and blood‐spinal cord barriers, pp. 117‐158 and H.S. Sharma (), Blood‐brain and spinal cord barriers in stress, pp. 231‐298, with permission from Elsevier.
  •   FigureFigure 11.14.3 Evans blue extravasation on the dorsal and ventral surfaces as well as in the deeper parts of the rat brain following heat stress. (A,B) Mild to moderate Evans blue staining of somatosensory cortex, pyriform cortex (pyr), cerebellum, hypothalamus (hypo), pons, and brainstem are evident. (CF) Coronal sections of rat brain from four different levels showing Evans blue extravasation in deep brain structures, such as (C) caudate‐putamen (cp) and (D‐F) hippocampus (hip), thalamus (thal), and hypothalamus. Abbreviations: acg, anterior cingulate cortex; fpm, frontal‐parietal motor area; fps, frontal‐parietal somatosensory area; pcg, posterior cingulate cortex; tem, temporal cortex. Coordinates from the bregma for coronal sections: C, +0.10 to +0.45; D, −3.25 to −3.90; E, −4.20 to −4.60; F, −5.25 to −6.65. Scale bars: B and D, 5 mm. Reprinted from The Blood‐Spinal Cord and Brain Barriers in Health and Disease (H.S. Sharma and J. Westman, eds.), H.S. Sharma (), Blood‐brain and spinal cord barriers in stress, pp. 231‐298, with permission from Elsevier.
  •   FigureFigure 11.14.4 Extravasation of Evans blue following heat stress. (A) Representation of midsagittal section of the rat brain. Staining of cerebroventricular walls of the lateral ventricles, fourth ventricle, and median eminence is apparent. (B,C) Cross‐sections of the brain passing through the caudate‐putamen (+0.45 from bregma for B) and hippocampal (−3.25 from bregma for C) levels showed leakage of Evans blue in the primary somatosensory cortex, pyriform cortex, hippocampus, hypothalamus, and amygdala. Scale bar indicates 5 mm in A and 4 mm in B,C. Reprinted from The Blood‐Spinal Cord and Brain Barriers in Health and Disease (H.S. Sharma and J. Westman, eds.), H.S. Sharma (), Blood‐brain and spinal cord barriers in stress, pp. 231‐298, with permission from Elsevier.
  •   FigureFigure 11.14.5 Changes in (A) rectal temperature and (B) brain edema formation in acute and chronic heat‐exposed rats. Heat‐adapted (HA) rats were exposed for 1 hr at 38°C for 7 days before the start of this experiment. A significant increase in rectal temperature was seen following 1‐, 2‐, and 4‐hr heat exposure in the naive and HA rats, although the hyperthermia was milder in the HA rats. Brain water content increased significantly after 4 hr of heat stress (HS) at 38°C in the naive but not the HA rats. When the adapted rats were subjected to an additional 4 hr of heat exposure on the second day (4 hr + 4 hr), however, profound hyperthermia and edema formation can be seen. Values are mean ± SD from six to eight rats. * P < 0.05, ** P < 0.01; ANOVA followed by Dunnet's test for multiple group comparison with one control group. Adapted from Sharma et al. (, ).

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Internet Resources
   http://www.temperatures.com/thermivendors.html
  A resource for thermistor probes, with links to various suppliers.
   http://www.myneurolab.com/
  Provides purchasing information for a 3‐mm thermistor probe suitable for rats.
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