Merry C. Nishimura

Rat middle cerebral artery occlusion: evaluation of the model and development of a neurologic examination.

We have examined the incidence and size of infarction after occlusion of different portions of the rat middle cerebral artery (MCA) in order to define the reliability and predictability of this model of brain ischemia. We developed a neurologic examination and have correlated changes in neurologic status with the size and location of areas of infarction. The MCA was surgically occluded at different sites in six groups of normal rats. After 24 hr, rats were evaluated for the extent of neurologic deficits and graded as having severe, moderate, or no deficit using a new examination developed for this model. After rats were sacrificed the incidence of infarction was determined at histologic examination. In a subset of rats, the size of the area of infarction was measured as a percent of the area of a standard coronal section. Focal (1-2 mm) occlusion of the MCA at its origin, at the olfactory tract, or lateral to the inferior cerebral vein produced infarction in 13%, 67%, and 0% of rats, respectively (N = 38) and produced variable neurologic deficits. However, more extensive (3 or 6 mm) occlusion of the MCA beginning proximal to the olfactory tract--thus isolating lenticulostriate end-arteries from the proximal and distal supply--produced infarctions of uniform size, location, and with severe neurologic deficit (Grade 2) in 100% of rats (N = 17). Neurologic deficit correlated significantly with the size of the infarcted area (Grade 2, N = 17, 28 +/- 5% infarction; Grade 1, N = 5, 19 +/- 5%; Grade 0, N = 3, 10 +/- 2%; p less than 0.05). We have characterized precise anatomical sites of the MCA that when surgically occluded reliably produce uniform cerebral infarction in rats, and have developed a neurologic grading system that can be used to evaluate the effects of cerebral ischemia rapidly and accurately. The model will be useful for experimental assessment of new therapies for irreversible cerebral ischemia.

Evaluation of 2,3,5-triphenyltetrazolium chloride as a stain for detection and quantification of experimental cerebral infarction in rats.

We have evaluated the use of 2,3,5-triphenyltetrazolium chloride (TTC) as an histopathologic stain for identification of infarcted rat brain tissue. The middle cerebral artery (MCA) of 35 normal adult rats was occluded surgically. At various times after surgical occlusion, rats were sacrificed and brain slices were obtained and stained with TTC or hematoxolin and eosin (H & E); the size of the area of infarcted tissue stained by each method was quantified. In rats sacrificed 24 hr after occlusion of the MCA, the size of the area of infarction was 21 +/- 2% of the coronal section for TTC, and 21 +/- 2% for H & E (mean +/- S.D., N = 13). The size of areas of infarction determined by either staining method was not significantly different in area by the paired test, and a significant correlation between sizes determined by each method was found by linear regression analysis (r = 0.91, slope = 0.89, and the y intercept = 4.4%). Staining with TTC is a rapid, convenient, inexpensive, and reliable method for the detection and quantification of cerebral infarction in rats 24 hr after the onset of ischemia.

Evaluation of 2,3,5-triphenyltetrazolium chloride staining to delineate rat brain infarcts.

Background and Purpose: Accurate and reproducible determination of the size and location of cerebral infarcts is critical for the evaluation of experimental focal cerebral ischemia. The purpose of this study was to compare intracardiac perfusion of 2,3,5-triphenyltetrazolium chloride with immersion of brain tissue in 2,3,5-triphenyltetrazolium chloride to delineate brain infarcts in rats. Methods: After 6, 24, or 48 hours of ischemia induced by permanent middle cerebral artery occlusion, some rats were perfused with 2,3,5-triphenyltetrazolium chloride; other rats were given an overdose of barbiturates, after which brain sections were immersed in 2,3,5- triphenyltetrazolium chloride. Coronal sections were taken 4, 6, and 8 mm from the frontal pole, and infarct areas in perfused and immersed sections were compared; subsequently, the same sections were stained with hematoxylin and eosin. Results: In rats subjected to 24 or 48 hours of occlusion, areas of infarction were clearly defined with both 2,3,5-triphenyltetrazolium chloride staining techniques, and the infarct sizes correlated well with the results of hematoxylin and eosin staining (r=0.85−0.94). Conclusions: These results demonstrate that intracardiac perfusion of 2,3,5-triphenyltetrazolium chloride is an accurate, inexpensive, and efficient staining method to detect infarcted tissue 24 and 48 hours after the onset of ischemia in rats.

The Effect of Hypoxia on Traumatic Head Injury in Rats: Alterations in Neurologic Function, Brain Edema, and Cerebral Blood Flow

We evaluated the effects of early posttraumatic hypoxia on neurologic function, magnetic resonance images (MRI), brain tissue specific gravities, and cerebral blood flow (CBF) in head-injured rats. By itself, an hypoxic insult (PaO2 40 mm Hg for 30 min) had little effect on any measure of cerebral function. After temporal fluid-percussion impact injury, however, hypoxia significantly increased morbidity. Of rats subjected to impact (4.9 ± 0.3 atm) plus hypoxia, 71% had motor weakness contralateral to the impact side 24 h after injury, while only 29% of rats subjected to impact alone had demonstrable weakness (p < 0.05). Lesions observed on MR images 24 h after injury were restricted to the impact site in rats with impact injury alone, but extensive areas with longer T1 relaxation times were observed throughout the ipsilateral cortex in rats with impact injury and hypoxic insult. Brain tissue specific gravity measurements indicated that much more widespread and severe edema developed in rats with impact injury and hypoxia. [14C]Iodoantipyrine autoradiography performed 24 h after injury showed that there was extensive hypoperfusion of the entire ipsilateral cortex in rats with impact injury and hypoxia. These results show that large areas of impact-injured brain are extremely vulnerable to secondary insults that can irreparably damage neural tissue, and provide experimental evidence for the observed adverse effects of hypoxia on outcome after human head injury.

Effect of hypoxia on traumatic brain injury in rats: Part 1. Changes in neurological function, electroencephalograms, and histopathology.

The effect of hypoxia on neurological function, compressed spectral array electroencephalography, and histopathology in head-injured rats was evaluated. By itself, an hypoxic insult (PaO2, 40 mm Hg for 30 minutes) caused no neurological deficit. Twenty per cent of rats injured by a 5-atmosphere temporal fluid percussion impact were hemiparetic contralateral to the side of impact, whereas 80% had no deficit 24 hours after injury. Seventy per cent of rats with both fluid impact injury and hypoxic insult, however, either had a definite hemiparesis, showed no spontaneous movement, or died (P less than 0.02). Impact alone produced an initial depression in electroencephalogram power that was prolonged in rats with hypoxic insult; the most dramatic effect was found in the injured hemisphere, with shorter and less profound effects in the contralateral hemisphere. Perfusion staining of injured cerebral tissue in vivo with 2,3,5-triphenyltetrazolium chloride showed an area of extensive ischemia around the impact site in rats with hypoxic insult. This ischemic area was not present in rats with either impact injury or hypoxia alone. We conclude that posttraumatic hypoxia clearly increases the severity of impact injury in this rat model. These findings suggest that hypoxia, which is common in head-injured patients, very likely worsens the effect of impact injury and may account for much of the diffuse neurological dysfunction in patients with severe craniocerebral trauma.

Effect of hypoxia on traumatic brain injury in rats: Part 2. Changes in high energy phosphate metabolism.

The effect of different degrees of hypoxia on phosphate metabolism in the brains of impact-injured rats was studied using in vivo phosphorus-31 magnetic resonance (P-31 MR) spectroscopy. Sequential changes in P-31 MR spectra within 60 minutes of insult were compared among rats with hypoxia alone, impact injury alone, or a combined impact-hypoxic insult. Hypoxia alone (PaO2 of 40 mm Hg for 30 minutes) caused no remarkable changes in phosphorus spectra except a decrease in intracellular pH. In impact-injured rats, the concentration of inorganic phosphate (Pi) increased, but signals for phosphocreatine (PCr) and beta-adenosine triphosphate (beta-ATP) did not change, and the ratio of PCr/Pi changed only slightly to 7% below control value. When rats with a fluid percussion impact injury of 5 atm were subjected to hypoxic conditions of a PaO2 of 40 mm Hg for 15 minutes, the PCr/Pi ratio decreased by 14%, a value significantly below that of the impact alone group (P less than 0.05). After longer periods of hypoxia (PaO2 of 40 mm Hg for 30 minutes) in impact-injured rats, there were marked increases of Pi and significant decreases in signals for PCr and beta-ATP, which caused a marked decrease in the PCr/Pi ratio to 39% below control values (P less than 0.001). Milder hypoxia (PaO2 of 50 mm Hg for 30 minutes) plus impact injury caused smaller changes in high energy metabolite concentrations, and the PCr/Pi ratio decreased to 15% below control values.(ABSTRACT TRUNCATED AT 250 WORDS)

Pentobarbital protection from cerebral infarction without suppression of edema.

We studied the mechanism of barbiturate protection from focal cerebral infarction in cats by examining in detail edema formation 72 hours after acute, permanent occlusion of the left middle cerebral artery (LMCA). Neurological function, gas exchange, vital signs, and intracranial pressure (ICP) were observed during the post-occlusion period, and infarct size and cerebral edema were measured after sacrifice. Infarct size was reduced only when pentobarbital was given before occlusion and continued for 24 hours. Edema formation was not suppressed even though the extent of infarction was. Clinical evidence of stroke developed and ICP rose in most cats after occlusion despite the presence of pentobarbital sufficient to reduce infarct size. Elevated ICP accounted for most premature deaths despite intensive cardiopulmonary support. Water and electrolyte changes in the ischemic hemisphere continued to develop throughout the 72 hour post-occlusion period in pentobarbital-treated cats, suggesting that resolution of edema was delayed by the drug. We conclude that pentobarbital reduces infarct size and attenuates the expected time course of ischemic edema in cats, but that the drug has little effect on the severity of edema that develops after arterial occlusion.

The Role of Ischemia in the Pathogenesis of Cervical Spondylotic Myelopathy: A Review and New Microangiographic Evidence

The fundamental cause of cervical spondylotic myelopathy is discussed in terms of anatomic, pathologic, clinical, and experimental evidence. The authors conclude that compression and ischemia of the spinal cord are additive causes of myelopathy in man. Neurologic and microangiographic data from 38 dogs with experimental cervical myelopathy are presented to substantiate the concept of additive etiologic factors.

In vivo sodium-23 magnetic resonance surface coil imaging: Observing experimental cerebral ischemia in the rat

Sodium-23 magnetic resonance imaging can be used to detect and assess experimental cerebral ischemia in the rat. An imaging technique utilizing a surface coil is described to produce sodium magnetic resonance images of good quality and resolution within 10 min. A novel method of hemispheric occlusion showed edema in the right brain of the rat head within 3 hr after injury. The edema was especially pronounced by 12 hr with effects in the right brain, eye and surrounding muscle evident.

Proton nuclear magnetic resonance spectroscopy of normal and edematous brain tissue in vitro: Changes in relaxation during tissue storage

Proton nuclear magnetic resonance relaxation times, T1 and T2, of water in unfixed gray and white matter from normal and edematous rabbit brain tissues were measured in vitro at 23 degrees C and 100 MHz to evaluate the effects of the temperature (-25 degrees C to 37 degrees C) and duration (0 to 96 h) of tissue storage on relaxation times. T1 and T2 tended to decrease during storage, probably from slow dehydration of the tissue. This effect was greatest in tissues stored at 37 degrees C and least in those stored at 4 and -25 degrees C; decreases in T1 and T2 were greater in white matter than in gray matter. Freezing brain tissue to -25 degrees C caused a sudden decrease in the T2 of normal white matter. Relaxation times were constant for 5 h in tissues stored at 23 degrees C and for 40 h at 4 degrees C. These results correlated well with corresponding tissue water loss.