Desmethyl tirilazad improves neurologic function after hypoxic ischemic brain injury in piglets.

OBJECTIVE: Desmethyl tirilazad is a lipid-soluble free radical quencher. Deferoxamine reduces free radicals by chelating iron and reducing hydroxyl formation. Free radical inhibitors have shown promise in several hypoxic ischemic brain injury models, and we wished to see if this work could be extended to our newborn piglet model. DESIGN: Randomized controlled trial. SUBJECTS: Piglets (0 to 3 days old). INTERVENTION: Carotid snares and arterial and venous catheters were placed under 1.5% isoflurane anesthesia. In Experiment 1, piglets were randomly assigned to receive either 3 mg/kg desmethyl tirilazad or vehicle at -15 and 90 mins. In Experiment 2, piglets were randomly assigned to receive either 20 mg/kg desmethyl tirilazad at -15 mins followed by 8 mg/kg/hr for 90 mins or 100 mg/kg deferoxamine at -15 mins or vehicle. At time 0, both carotid arteries were clamped and blood was withdrawn to reduce the blood pressure to two-thirds normal. At 15 mins, inspired oxygen was reduced to 6%. At 30 mins, the carotid snares were released, the withdrawn blood was reinfused, and the oxygen was switched to 100%. On the third day after the hypoxic ischemic injury, the animals were killed by perfusing their brains with 10% formalin. We tested the timing of lipid peroxidation and inhibition of lipid peroxidation by these agents by freezing the brains of a subset of pigs in liquid nitrogen. MEASUREMENTS: Neurologic examination and brain pathology were scored by blinded observers. Thiobarbituric acid-reactive substance and oxidized and reduced glutathione were measured on frozen brains. MAIN RESULTS: Desmethyl tirilazad (20 mg/kg) and 100 mg/kg deferoxamine inhibit lipid peroxidation. Desmethyl tirilazad (20 mg/kg) improves neurologic exam, but 3 mg/kg Desmethyl tirilazad or 100 mg/kg deferoxamine does not. Neither desmethyl tirilazad nor deferoxamine improves pathologic results. CONCLUSIONS: High-dose desmethyl tirilazad improves neurologic function after hypoxic ischemic brain injury in the newborn piglet.

The effect of glucose during ischemia on brain ATP, lactate, and glutamate in piglets.

Glucose worsens hypoxic-ischemic brain injury in 0- to 3-day-old piglets. Piglets were randomly assigned to have blood glucose increased with glucose infusion (n = 12), or decreased with insulin (n = 13), or a sham group (n = 10). In the insulin and glucose groups at time 0, both carotid arteries were clamped, and blood was withdrawn to reduce the blood pressure one third. At time 15 min FiO2 was reduced to 6%. At time 30 min cerebrospinal fluid (CSF) was taken for glutamate, and the brains were frozen. The shams had CSF removed and brains frozen without hypoxia or ischemia. Brain ATP was 1.7 +/- 0.09 mumol/g wet weight in the shams, 0.98 +/- 0.09 in the glucose group (p < 0.01 vs. sham), and 0.52 +/- 0.10 in the insulin group (p < 0.01 vs. glucose). Brain lactate levels were 4.3 +/- 1.0 mumol/g wet weight in the shams, 18.3 +/- 1.9 in the insulin group (p < 0.01 vs. sham), and 29.4 +/- 2.6 in the glucose group (p < 0.01 vs. insulin). CSF glutamate was 9.3 +/- 3.6 microM in the glucose group, 9.6 +/- 3.5 in the insulin group, and 2.2 +/- 0.9 in the shams (p < 0.05, glucose and insulin > sham). The Bmax for MK-801 binding was 2.3 +/- 0.2 pmol/mg protein in the glucose group, 2.6 +/- 0.1 in the insulin group (p < 0.05 vs. sham), and 2.0 +/- 0.2 in the shams, and the Kd was the same in the three groups (p = nonsignificant). Brain Na+,K(+)-ATPase was the same in the three groups (p = nonsignificant). Providing additional glucose preserves ATP during hypoxic-ischemic brain injury in the piglet but does not change CSF glutamate or brain-801 binding and probably worsens outcome by elevating brain lactate levels above the threshold for cellular injury.

Adjunctive brainstem behaviors in the rat.

UNLABELLED: Adjunctive behaviors refers to behaviors that are "out of context" and/or excessive in amount. The terminology was first used for unexplained behaviors associated with food pellet ratio contingencies in food deprived rats (Falk, 1971). Clinically, unrelated and complex excessive behaviors were also noted with brainstem and diencephalic discharges (Andy, 1986, 1989 and 1995; Andy and Jurko, 1986). The objective of this study was to develop a model of brainstem generated adjunctive behaviors in the rat. METHOD: Sixty-one adult rats were used. Kindling was induced by mechanical and DC electrical perturbations in the midbrain. Nine different treatment groups were established in which lesions and drugs were used separately and in different combinations. Cocaine was used to accentuate, and dextrorphan to attenuate the kindling process. Behaviors were visually analyzed and recorded by video camera; they were not quantified. DC stimulation and EEGs were doen with Grass instruments. Recording and stimulation sights were verified histologically. RESULTS: A total of 45 different behaviors were elicited. DC stimulation kindled subjects had significantly more behaviors than did electrode insertion alone and cocaine alone. However, cocaine combined with DC stimulation increased the number and intensity of the kindled behaviors. Dextrorphan tended to facilitate catatonic and sleep states. Behaviors occurred randomly and were unrelated, even when they occurred in clusters. The behaviors were associated with four abnormal EEG discharge patterns. CONCLUSION: We speculate that brainstem behaviors are innate adjunctive drives that are generated in the brainstem reticular formation. They are monitored by the cortex and partially modified by environmental and cortical inputs.

Effects of neonatal exposure to anti-nerve growth factor on the number and size distribution of trigeminal neurones projecting to the molar dental pulp in rats.

The first aim of the present study was to determine whether depletion of endogenous nerve growth factor (NGF) during early postnatal development results in a long-term deficit in the number of trigeminal ganglion cells and axons projecting to the molar pulp. The second aim was to identify selectivity of the effects of NGF deprivation for any specific size group among pulp neurones. Newborn Sprague-Dawley rats were given subcutaneous injections of either rabbit anti-mouse-NGF serum or non-immune (control) rabbit serum for a period of 1 month. At age 4 months, Fluoro-gold (FG) was applied to the pulp chamber of the right maxillary first molar. One week later the animals were perfusion-fixed, and the trigeminal ganglia were removed and serially sectioned with a cryostat. Labelled neurones were seen only in the trigeminal ganglia ipsilateral to the injected teeth. The area of every labelled cell profile was measured, and from these data, estimates of the true number and size distribution of FG-labelled cells were obtained by recursive translation. Ganglia of control animals had a mean of 197 labelled neurones, all in the maxillary division, and most of the somas were of medium or large diameter. NGF-deprived animals had significantly fewer (mean = 145) FG-labelled cells in the trigeminal ganglion ipsilateral to the injected tooth. Neurones with somas of less than 30 microns dia were most strikingly subnormal in anti-NGF treated animals (64% of controls). In accordance with the greater susceptibility of small neurones to anti-NGF exposure, deficits in apical nerve fibres of the mandibular first molar were greater in degree and duration for unmyelinated axons than for myelinated axons. It is concluded that NGF is an important mediator in regulation of postnatal development of the sensory innervation of the dental pulp. The results also indicate that postnatal development of at least one class of larger pulpal afferent neurones is regulated by factors other than NGF.

The effects of anti-nerve growth factor on retrograde labelling of superior cervical ganglion neurones projecting to the molar pulp in the rat.

The aims were to demonstrate sympathetic ganglion neurones projecting to the rat molar pulp and to determine whether deprivation of nerve growth factor (NGF) in neonatal rats eliminates this source of pulpal innervation. Newborn Sprague-Dawley rats were given subcutaneous injections of rabbit anti-mouse-NGF serum for 1 month. Control animals included litter mates treated with preimmune serum and untreated, age-matched rats. AT 4 months of age, Fluoro-gold (FG) was applied to the pulp chamber of the right first maxillary molar. One week later, the animals were perfusion fixed, and the superior cervical ganglia (SCG) were removed, embedded in paraffin, and serially sectioned at 10 microns. FG-labelled cells were detected by epifluorescence microscopy with a u.v. filter set. Control animals had 5-10 FG-labelled neurones widely distributed throughout the SCG ipsilateral to the injection site and no labelled cells in the contralateral SCG. NGF-deprived animals had either no FG-labelled cells or a single labelled cell in the ipsilateral SCG. These results indicate that, in rats, (1) the number of SCG neurones projecting to the molar pulp is rather low, (2) SCG neurones that innervate the dental pulp of the maxillary molar pulp are dispersed throughout the ganglion, (3) the projection from SCG to the molar is exclusively ipsilateral, and (4) neonatal NGF deprivation induces a permanent, almost total, loss of sympathetic neurones projecting to the dental pulp.

Effects of postnatal anti-nerve growth factor serum exposure on development of apical nerves of the rat molar.

The present study was undertaken to test the hypothesis that development of the pulpal innervation is dependent on nerve growth factor (NGF). Newborn rats were given subcutaneous injections of a rabbit anti-mouse NGF serum on alternate days for the first 24 days postnatally. Control animals were untreated and normal rabbit serum-treated litter mates. The animals were deeply anesthetized on postnatal day 26, perfused with fixative and the first mandibular molars were processed for transmission electron microscopy to obtain a complete census of axons entering the four roots. The composition of the mental nerve was also examined. Compared to control animals, the apical innervation of molars from anti-NGF-treated rats had only 62% as many myelinated fibers and 41% as many unmyelinated axons. Those myelinated fibers present in antiserum-treated animals were slightly, but significantly, smaller in average diameter than controls. In teeth of control animals, about 20% of all unmyelinated axons were located in fibers coursing outside of nerve fascicles; these isolated fibers were disproportionately rare after antiserum exposure. The average number of unmyelinated axons per Schwann cell unit was also significantly lower. Postnatal exposure to anti-NGF had milder effects on mental nerve composition compared to the tooth innervation. Numbers of myelinated fibers were 83% of controls, unmyelinated axons were 74% of controls and there was no change in the average number of unmyelinated axons per Schwann cell unit. We conclude that development of dental innervation is highly susceptible to postnatal NGF deprivation. This may be a consequence of the mostly nociceptive composition of dental nerves and their late development.

Quantitative study of the apical nerve fibers of adult and juvenile rat molars.

The rat molar has become an important model for studies of interactions between nerves and the pulp-dentin complex, yet there is only limited quantitative information on the number and size distribution of axons entering the roots of this tooth. This study was undertaken to provide such a detailed characterization of the apical innervation of the rat molar. An additional objective was to compare the apical nerve composition of young, recently erupted rat molars with that of mature teeth in order to determine whether there is ongoing maturation of the innervation after the teeth have attained functional occlusion. A complete census was made of the nerve fibers entering the roots of both mature and recently erupted juvenile mandibular first molars in Sprague-Dawley rats. Each of the four roots of the first molars was processed for electron microscopy of thin sections near the apex. The majority of intradental nerve fibers entered the molar via the two larger (mesial and distal) roots. Within the apical root pulp, most, but not all, axons occurred within well-defined fascicles associated with blood vessels. Molars from adult animals (age 4 months) had a mean total of 232 (S.D. = 49, N = 7 teeth) myelinated fibers and 806 (S.D. = 143) unmyelinated axons entering the four roots. Fibers exceeding the A delta size range (circumference > or = 19 microns) accounted for only 4% of the myelinated axons at the apex. Molars from juvenile animals (age 4 weeks) had fewer myelinated fibers (mean 176, S.D. 18, N = 8), but more unmyelinated axons (mean 1,174, S.D. 160) than adults. The mean ratio of unmyelinated axons to myelinated axons was 6.6:1 for juveniles compared to 3.5:1 for adults. Juvenile teeth contained no myelinated fibers that exceeded 19 microns in circumference. These results indicate that the innervation of the rat molar resembles that of teeth of non-rodent mammals in that (1) innervation density is high, (2) there is a high ratio of unmyelinated axons, and (3) most of the myelinated fibers are of thin caliber. Furthermore, it appears that after the molar erupts, maturation of the nerve fiber composition continues with processes that include both a marked decrease in the number of unmyelinated axons and an increase in the number and size heterogeneity of myelinated fibers.

Cocaine-induced brainstem seizures and behavior.

A variety of abnormal sensory/motor behaviors associated with electrical discharges recorded from the bilateral brainstem were induced in adult WKY rats by mechanical (electrode implants) and DC electrical current stimulations and by acute and chronic administration of cocaine. The electrode implant implicated one side or the other of the reticular system of the brainstem but subjects were not incapacitated by the stimulations. Cocaine (40 mg/kg) was injected subcutaneously for an acute experiment and subsequent 20 mg/kg doses twice daily for 3 days in a chronic study. Cocaine generated more abnormal behaviors in the brainstem perturbation group, especially the electrically perturbated subjects. The abnormal behaviors were yawning, retrocollis, hyperactivity, hypersensitivity, "beating drum" behavior, squealing, head bobbing, circling, sniffing, abnormal posturing, and facial twitching. Shifts in the power frequency spectra of the discharge patterns were noted between quiet and pacing behavioral states. Hypersensitivity to various auditory, tactile, and visual stimulation was present and shifts in the brainstem ambient power spectral frequency occurred in response to tactile stimulation. These findings suggest that the brainstem generates and propagates pathological discharges that can be elicited by mechanical and DC electrical perturbation. Cocaine was found to activate the discharge system and thus induce abnormal behaviors that are generated at the discharge site and at distant sites to which the discharge propagates. Cognitive functions may also be involved since dopaminergic and serotonergic cellular elements at the brainstem level are also implicated.