Robert H. Garman

Blast exposure in rats with body shielding is characterized primarily by diffuse axonal injury.

Blast-induced traumatic brain injury (TBI) is the signature insult in combat casualty care. Survival with neurological damage from otherwise lethal blast exposures has become possible with body armor use. We characterized the neuropathologic alterations produced by a single blast exposure in rats using a helium-driven shock tube to generate a nominal exposure of 35 pounds per square inch (PSI) (positive phase duration ∼ 4 msec). Using an IACUC-approved protocol, isoflurane-anesthetized rats were placed in a steel wedge (to shield the body) 7 feet inside the end of the tube. The left side faced the blast wave (with head-only exposure); the wedge apex focused a Mach stem onto the rats head. The insult produced ∼ 25% mortality (due to impact apnea). Surviving and sham rats were perfusion-fixed at 24 h, 72 h, or 2 weeks post-blast. Neuropathologic evaluations were performed utilizing hematoxylin and eosin, amino cupric silver, and a variety of immunohistochemical stains for amyloid precursor protein (APP), glial fibrillary acidic protein (GFAP), ionized calcium-binding adapter molecule 1 (Iba1), ED1, and rat IgG. Multifocal axonal degeneration, as evidenced by staining with amino cupric silver, was present in all blast-exposed rats at all time points. Deep cerebellar and brainstem white matter tracts were most heavily stained with amino cupric silver, with the morphologic staining patterns suggesting a process of diffuse axonal injury. Silver-stained sections revealed mild multifocal neuronal death at 24 h and 72 h. GFAP, ED1, and Iba1 staining were not prominently increased, although small numbers of reactive microglia were seen within areas of neuronal death. Increased blood-brain barrier permeability (as measured by IgG staining) was seen at 24 h and primarily affected the contralateral cortex. Axonal injury was the most prominent feature during the initial 2 weeks following blast exposure, although degeneration of other neuronal processes was also present. Strikingly, silver staining revealed otherwise undetected abnormalities, and therefore represents a recommended outcome measure in future studies of blast TBI.

Adenosine A1 receptor knockout mice develop lethal status epilepticus after experimental traumatic brain injury.

Adenosine, acting at A1 receptors, exhibits anticonvulsant effects in experimental epilepsy—and inhibits progression to status epilepticus (SE). Seizures after traumatic brain injury (TBI) may contribute to pathophysiology. Thus, we hypothesized that endogenous adenosine, acting via A1 receptors, mediates antiepileptic benefit after experimental TBI. We subjected A1-receptor knockout (ko) mice, heterozygotes, and wild-type (wt) littermates (n = 115) to controlled cortical impact (CCI). We used four outcome protocols in male mice: (1) observation for seizures, SE, and mortality in the initial 2 h, (2) assessment of seizure score (electroencephalogram (EEG)) in the initial 2 h, (3) assessment of mortality at 24 h across injury levels, and (4) serial assessment of arterial blood pressure, heart rate, blood gases, and hematocrit. Lastly, to assess the influence of gender on this observation, we observed female mice for seizures, SE, and mortality in the initial 2 h. Seizure activity was noted in 83% of male ko mice in the initial 2 h, but was seen in no heterozygotes and only 33% of wt (P < 0.05). Seizures in wt were brief (1 to 2 secs). In contrast, SE involving lethal sustained (>1 h) tonic clonic activity was uniquely seen in ko mice after CCI (50% incidence in males), (P < 0.05). Seizure score was twofold higher in ko mice after CCI versus either heterozygote or wt (P < 0.05). An injury-intensity dose–response for 24 h mortality was seen in ko mice (P < 0.05). Physiologic parameters were similar between genotypes. Seizures were seen in 100% of female ko mice after CCI versus 14% of heterozygotes and 25% wt (P < 0.05) and SE was restricted to the ko mice (83% incidence). Our data suggest a critical endogenous anticonvulsant action of adenosine at A1 receptors early after experimental TBI.

Delayed, spontaneous hypothermia reduces neuronal damage after asphyxial cardiac arrest in rats.

ObjectiveCore temperature is reduced spontaneously after asphyxial cardiac arrest in rats. To determine whether spontaneous hypothermia influences neurologic damage after asphyxial arrest, we compared neurologic outcome in rats permitted to develop spontaneous hypothermia vs. rats managed with controlled normothermia. InterventionsMale Sprague-Dawley rats were asphyxiated for 8 mins and resuscitated. After extubation, a cohort of rats was managed with controlled normothermia (CN) by placement in a servo-controlled incubator set to maintain rectal temperature at 37.4°C for 48 hrs. CN rats were compared with permissive hypothermia (PH) rats that were returned to an ambient temperature environment after extubation. Rats were killed at either 72 hrs (PH72hr, n = 14; CN72hr, n = 9) or 6 wks (PH6wk, n = 6, CN6wk, n = 6) after resuscitation. PH72 rats were historic controls for the CN72 rats, whereas PH6 and CN6 rats were randomized and studied contemporaneously. MeasurementsA clinical neurodeficit score (NDS) was determined daily. A pathologist blinded to group scored 40 hematoxylin and eosin -stained brain regions for damage by using a 5-point scale (0 = none, 5 = severe). Quantitative analysis of CA1 hippocampus injury was performed by counting normal-appearing neurons in a defined subsection of CA1. Main ResultsMean rectal temperatures measured in the PH6wk rats (n = 6) were 36.9, 34.8, 35.5, 36.7, and 37.4°C at 2, 8, 12, 24, and 36 hrs, respectively. Mortality rate (before termination) was lower in PH compared with CN (0/20 vs. 7/15;p < .005). PH demonstrated a more favorable progression of NDS (p = .04) and less weight loss (p < .005) compared with CN. Median histopathology scores were lower (less damage) in PH72hr vs. CN72hr for temporal cortex (0 vs. 2.5), parietal cortex (0 vs. 2), thalamus (0 vs. 3), CA1 hippocampus (1.5 vs. 4.5), CA2 hippocampus (0 vs. 3.5), subiculum (0 vs. 4), and cerebellar Purkinje cell layer (2 vs. 4) (all p < .05). There was almost complete loss of normal-appearing CA1 neurons in CN72hr rats (6 ± 2 [mean ± sd] normal neurons compared with 109 ± 12 in naïve controls). In contrast, PH72hr rats demonstrated marked protection (97 ± 23 normal-appearing neurons) that was still evident, although attenuated, at 6 wks (42 ± 24 normal-appearing neurons, PH6wk). ConclusionRats resuscitated from asphyxial cardiac arrest develop delayed, mild to moderate, prolonged hypothermia that is neuroprotective.

Histology of the Central Nervous System

The intent of this article is to assist pathologists inexperienced in examining central nervous system (CNS) sections to recognize normal and abnormal cell types as well as some common artifacts. Dark neurons are the most common histologic artifact but, with experience, can readily be distinguished from degenerating (eosinophilic) neurons. Neuron degeneration stains can be useful in lowering the threshold for detecting neuron degeneration as well as for revealing degeneration within populations of neurons that are too small to show the associated eosinophilic cytoplasmic alteration within H&E-stained sections. Neuron degeneration may also be identified by the presence of associated macroglial and microglial reactions. Knowledge of the distribution of astrocyte cytoplasmic processes is helpful in determining that certain patterns of treatment-related neuropil vacuolation (as well as some artifacts) represent swelling of these processes. On the other hand, vacuoles with different distribution patterns may represent alterations of the myelin sheath. Because brains are typically undersampled for microscopic evaluation, many pathologists are unfamiliar with the circumventricuar organs (CVOs) that represent normal brain structures but are often mistaken for lesions. Therefore, the six CVOs found in the brain are also illustrated in this article.

Induced hyperthermia exacerbates neurologic neuronal histologic damage after asphyxial cardiac arrest in rats.

BackgroundTemperature is an important modulator of the evolution of ischemic brain injury—with hypothermia lessening and hyperthermia exacerbating damage. We recently reported that children resuscitated from predominantly asphyxial arrest often develop an initial spontaneous hypothermia followed by delayed hyperthermia. The initial hypothermia observed in these children was frequently treated with warming lights which, despite careful monitoring, often resulted in overshoot hyperthermia. We have previously reported in a rat model of asphyxial cardiac arrest that active warming, to prevent spontaneous hypothermia, worsens brain injury. ObjectiveWe sought to determine whether delayed induction of hyperthermia would worsen brain injury after asphyxial arrest in rats. DesignMale Sprague-Dawley rats were asphyxiated for 8 mins and resuscitated. An implantable temperature probe was placed into the peritoneum before asphyxia. The probe is a component of a computer-based, radiofrequency, telemetry system (Minimitter, Sunriver, OR) that allowed continuous acquisition and manipulation (via heating and cooling devices) of core (intraperitoneal) body temperature. Body temperature was monitored but not manipulated for the first 24 hrs of recovery. Rats were assigned to: no temperature manipulation (n = 21), induced hyperthermia (40 ± 0.5°C) for 3 hrs beginning at 24 hrs (n = 21), or induced hyperthermia at 48 hrs (n = 10). Control groups included sham rats (all surgical procedures except asphyxia) treated with induced hyperthermia at 24 hrs (n = 4) or 48 hrs (n = 4) and naïve rats (n = 4). Rats were killed at 7 days and injured neurons in hematoxylin and eosin stained coronal brain sections through dorsal hippocampus were scored in a semiquantitative manner on a scale of 0 to 10 (0 = normal; 1 = up to 10% neurons with ischemic neuronal changes; 10 = 90–100% neurons with ischemic neuronal changes). Normal-appearing neurons were also counted in CA1. The number of normal-appearing neurons in a 20× field in CA1 were also counted. Main ResultsAll naïve and sham hyperthermia control rats survived the protocol. There was a trend toward a larger mortality rate in asphyxiated rats treated with induced hyperthermia at 24 hrs (9 of 21 died) vs. asphyxiated rats without induced hyperthermia (3 of 21) or with hyperthermia induced at 48 hrs (3 of 10) (Kaplan-Meier p = .0595). Asphyxiated rats with hyperthermia induced at 24 hrs had larger (worse) histopathology damage scores than rats subjected to asphyxia without induced hyperthermia (9.3 ± 1.5 vs. 6.2 ± 2.6;p = .001). Histopathology damage scores in asphyxiated rats with hyperthermia induced at 48 hrs did not differ from those in rats asphyxiated without induced hyperthermia (6.4 ± 3.0 vs. 6.2 ± 2.6;p = .907). There were fewer normal-appearing CA1 neurons in asphyxiated rats with hyperthermia induced at 24 hrs vs. rats subjected to asphyxia without induced hyperthermia (33 ± 13 vs. 67 ± 36;p = .002). The number of normal-appearing CA1 neurons in asphyxiated rats with hyperthermia induced at 48 hrs did not differ from that in rats asphyxiated without induced hyperthermia (59 ± 21 vs. 67 ± 36;p = .885). ConclusionsInduced hyperthermia when administered at 24 hrs, but not 48 hrs, worsens ischemic brain injury in rats resuscitated from asphyxial cardiac arrest. This may have implications for postresuscitative management of children and adults resuscitated from cardiac arrest. The common clinical practice of actively warming patients with spontaneous hypothermia might result in iatrogenic injury if warming results in hyperthermic overshoot. Avoidance of hyperthermia induced by active warming at critical time periods after cardiac arrest may be important.

Developmental Neurotoxicity Study of Dietary Bisphenol A in Sprague-Dawley Rats

This study was conducted to determine the potential of bisphenol A (BPA) to induce functional and/or morphological effects to the nervous system of F1 offspring from dietary exposure during gestation and lactation according to the Organization for Economic Cooperation and Development and U.S. Environmental Protection Agency guidelines for the study of developmental neurotoxicity. BPA was offered to female Sprague-Dawley Crl:CD (SD) rats (24 per dose group) and their litters at dietary concentrations of 0 (control), 0.15, 1.5, 75, 750, and 2250 ppm daily from gestation day 0 through lactation day 21. F1 offspring were evaluated using the following tests: detailed clinical observations (postnatal days [PNDs] 4, 11, 21, 35, 45, and 60), auditory startle (PNDs 20 and 60), motor activity (PNDs 13, 17, 21, and 61), learning and memory using the Biel water maze (PNDs 22 and 62), and brain and nervous system neuropathology and brain morphometry (PNDs 21 and 72). For F1 offspring, there were no treatment-related neurobehavioral effects, nor was there evidence of neuropathology or effects on brain morphometry. Based on maternal and offspring body weight reductions, the no-observed-adverse-effect level (NOAEL) for systemic toxicity was 75 ppm (5.85 and 13.1 mg/kg/day during gestation and lactation, respectively), with no treatment-related effects at lower doses or nonmonotonic dose responses observed for any parameter. There was no evidence that BPA is a developmental neurotoxicant in rats, and the NOAEL for developmental neurotoxicity was 2250 ppm, the highest dose tested (164 and 410 mg/kg/day during gestation and lactation, respectively).

Practical Aspects of Neuropathology: A Technical Guide for Working with the Nervous System

Toxicologic pathologists are evaluating tissues from the central and peripheral nervous systems with increasing frequency. This change is being driven by recently established regulatory guidelines and intense interest in developing pharmaceutical compounds to treat various nervous system disorders. However, morphologic evaluation of the nervous system by light or electron microscopy requires special understanding and effort. Here, we review the general concepts of fixation for the nervous system, explain perfusion procedures for optimal preservation, and provide information on handling tissues to avoid artifacts. In general, fixation with aldehydes is recommended for nervous tissue (a combination of paraformaldehyde and glutaraldehyde is preferred). Electron microscopic studies require fixatives of the highest purity possible, typically paraformaldehyde prepared fresh from powder mixed with high-grade glutaraldehyde. The final osmolality of the solution should be slightly hypertonic, in the range of 400-600 mOsmol. Slight hypertonicity is very important and will facilitate maintenance of vascular distention during whole-body perfusion, which is the best method for producing high-quality tissue preparations. Special effort is necessary for handling nervous tissue in a way that minimizes artifacts because chemical fixation is not completed immediately following the perfusion. These technical details should help toxicologic pathologists in their efforts to work with the nervous system, thereby increasing their effectiveness in supporting safety characterization of new test materials undergoing toxicologic assessments.

A ‘Best Practices’ Approach to Neuropathologic Assessment in Developmental Neurotoxicity Testing—for Today:

A key trait of developmental neurotoxicants is their ability to cause structural lesions in the immature nervous system. Thus, neuropathologic assessment is an essential element of developmental neurotoxicity (DNT) studies that are designed to evaluate chemically-induced risk to neural substrates in young humans. The guidelines for conventional DNT assays have been established by regulatory agencies to provide a flexible scaffold for conducting such studies; recent experience has launched new efforts to update these recommendations. The present document was produced by an ad hoc subcommittee of the Society of Toxicologic Pathology (STP) tasked with examining conventional methods used in DNT neuropathology in order to define the ‘best practices’ for dealing with the diverse requirements of both national (EPA) and international (OECD) regulatory bodies. Recommendations (including citations for relevant neurobiological and technical references) address all aspects of the DNT neuropathology examination: study design; tissue fixation, collection, processing, and staining; qualitative and quantitative evaluation; statistical analysis; proper control materials; study documentation; and personnel training. If followed, these proposals will allow pathologists to meet the need for a sound risk assessment (balanced to address both regulatory issues and scientific considerations) in this field today while providing direction for the research needed to further refine DNT neuropathology ‘best practices’ in the future.

Hemorrhagic Shock after Experimental Traumatic Brain Injury in Mice: Effect on Neuronal Death

Traumatic brain injury (TBI) from blast injury is often complicated by hemorrhagic shock (HS) in victims of terrorist attacks. Most studies of HS after experimental TBI have focused on intracranial pressure; few have explored the effect of HS on neuronal death after TBI, and none have been done in mice. We hypothesized that neuronal death in CA1 hippocampus would be exacerbated by HS after experimental TBI. C57BL6J male mice were anesthetized with isoflurane, mean arterial blood pressure (MAP) was monitored, and controlled cortical impact (CCI) delivered to the left parietal cortex followed by continued anesthesia (CCI-only), or either 60 or 90 min of volume-controlled HS. Parallel 60- or 90-min HS-only groups were also studied. After HS (+/-CCI), 6% hetastarch was used targeting MAP of > or =50 mm Hg during a 30-min Pre-Hospital resuscitation phase. Then, shed blood was re-infused, and hetastarch was given targeting MAP of > or =60 mm Hg during a 30-min Definitive Care phase. Neurological injury was evaluated at 24 h (fluorojade C) or 7 days (CA1 and CA3 hippocampal neuron counts). HS reduced MAP to 30-40 mm Hg in all groups, p < 0.05 versus CCI-only. Ipsilateral CA1 neuron counts in the 90-min CCI+HS group were reduced at 16.5 +/- 14.1 versus 30.8 +/- 6.8, 32.3 +/- 7.6, 30.6 +/- 2.2, 28.1 +/- 2.2 neurons/100 mum in CCI-only, 60-min HS-only, 90-min HS-only, and 60-min CCI+HS, respectively, all p < 0.05. CA3 neuron counts did not differ between groups. Fluorojade C staining confirmed neurodegeneration in CA1 in the 90-min CCI+HS group. Our data suggest a critical time window for exacerbation of neuronal death by HS after CCI and may have implications for blast injury victims in austere environments where definitive management is delayed.

Opioid neurotoxicity: fentanyl dose-response effects in rats.

Opioids, when administered in large doses, produce brain damage, primarily in the limbic system and association areas in rats.This investigation examined the relationship between opioid dose and severity and frequency of brain damage in rats. Forty male Sprague-Dawley rats were anesthetized with halothane/N2 O and underwent tracheal intubation, mechanical ventilation, arterial/venous cannulation, and insertion of a rectal temperature probe and biparietal electroencephalogram electrodes. After surgery, halothane was discontinued and O2/N2 O 30%/70% was administered for 1 h. Rats were then randomly assigned to one of eight groups. The control group received a loading dose (LD) of 4 mL/kg of 0.9% normal saline solution (NSS) and a maintenance dose (MD) of 4 mL [centered dot] kg-1 [centered dot] h-1 NSS. The other groups were given fentanyl lypophilized and reconstituted in NSS with the LD ranging from 50 to 3200 micro g/kg and the MD from 2 to 128 micro g [centered dot] kg-1 [centered dot] min-1. After 2 h of fentanyl or NSS infusion, all rats received 100% O2 and, when alert, their tracheas were extubated; after 7 days the rats underwent cerebral perfusion fixation, followed by light microscopic evaluation. Histopathologic lesions (primarily eosinophilic neuron degeneration) were subjectively graded by a pathologist unaware of the experimental treatment; the grades were based on the percentage of dead neurons. There were no lesions observed in the brain areas in any of the control or 200-8 (LD, micro g/kg; MD, micro g [centered dot] kg-1 [centered dot] min-1) groups. Eleven of 20 rats in the 400-16, 800-32, 1600-64, and 3200-18 groups showed evidence of brain damage primarily in limbic system structures and association areas (P < 0.05). Our data confirm that fentanyl produces limbic system brain damage in rats, and that the damage occurs over a broad range of doses. (Anesth Analg 1996;83:1298-306)