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Chronic frontal lobe functional deficits after traumatic brain injury (TBI) may be associated with altered catecholamine systems in the frontal cortex. To test this, tyrosine hydroxylase (TH) and dopamine beta-hydroxylase (DBH) levels were examined by immunohistochemistry and Western blot at 1, 7, 14, and 28 days after TBI or sham surgery. No alterations in DBH levels were observed by Western blot at any time point examined, but there was a significant increase in TH expression 28 days after TBI (optical density 334 +/- 68% or 3.3-fold, ipsilateral and 218 +/- 39% or 2.2-fold, contralateral) relative to the sham controls. The increase in TH may reflect a compensatory response of dopaminergic neurons to upregulate their synthesizing capacity and increase the efficiency of dopamine neurotransmission chronically after TBI.

Experimental traumatic brain injury (TBI) produces cholinergic neurotransmission deficits that may contribute to chronic spatial memory deficits. Cholinergic neurotransmission deficits may result from presynaptic alterations in the storage and release of acetylcholine (ACh) or from changes in the receptors for ACh. The vesicular ACh transporter (VAChT) mediates accumulation of ACh into secretory vesicles, and the M2 muscarinic receptor subtype can modulate cholinergic neurotransmission via a presynaptic inhibitory feedback mechanism. We examined the effects of controlled cortical impact (CCI) injury on hippocampal VAChT and M2 muscarinic receptor subtype protein and medial septal mRNA levels at 4 weeks following injury. Rats were anesthetized and surgically prepared for CCI injury (4 m/sec, 2.5 to 2.9 mm in depth) and sham surgery. Animals were sacrificed, and coronal sections (35 microm thick) were cut through the dorsal hippocampus for VAChT and M2 immunohistochemistry. Semiquantitative measurements of VAChT and M2 protein in hippocampal homogenates from injured and sham rats were assessed with Western blot analysis. Changes in VAChT and M2 mRNA levels were evaluated by reverse transcriptase polymerase chain reaction (RT-PCR). At 4 weeks after injury, both immunohistochemical and Western blot methods demonstrated an increase in hippocampal VAChT protein. An increase in VAChT mRNA was also observed. Immunohistochemistry demonstrated a loss of M2; however, there was no significant change in M2 mRNA levels in comparison with sham controls. These changes may represent a compensatory response of cholinergic neurons to increase the efficiency of ACh neurotransmission chronically after TBI through differential transcriptional regulation.

Experimental traumatic brain injury (TBI) produces cholinergic neurotransmission deficits that may contribute to chronic spatial memory deficits. Cholinergic neurotransmission deficits may be due to presynaptic alterations in the storage and release of acetylcholine (ACh) or from changes in the receptors for ACh. The vesicular ACh transporter (VAChT) mediates accumulation of ACh into secretory vesicles, and M2 receptors can modulate cholinergic neurotransmission via a presynaptic inhibitory feedback mechanism. We examined the effects of controlled cortical impact (CCI) injury on hippocampal VAChT and M2 muscarinic subtype receptor protein levels at four time points: 1 day, 1 week, 2 weeks, and 4 weeks following injury. Rats were anesthetized and surgically prepared for controlled cortical impact injury (4 m/s, 2.5- to 2.9-mm depth) and sham surgery. Animals were sacrificed and coronal sections (35 micro(m) thick) were cut through the dorsal hippocampus for VAChT and M2 immunohistochemistry. Semiquantitative measurements of VAChT and M2 protein in hippocampal homogenates from injured and sham rats were assessed using Western blot analysis. Immunohistochemistry showed no obvious changes in VAChT and M2 immunoreactivity at 1 day and 1 week postinjury. At 2 and 4 weeks postinjury, an increase in hippocampal VAChT protein and a corresponding loss of hippocampal M2 protein was observed compared to sham controls. Consistent with these results, Western blot analyses at 4 weeks postinjury demonstrated a 40-50% increase in VAChT and a 25-30% decrease in M2. These changes may represent a compensatory response of cholinergic neurons to increase the efficiency of ACh neurotransmission chronically after TBI, by upregulating the storage capacity and subsequent release of ACh and downregulating presynaptic inhibitory receptors.

Photic evoked responses were recorded from the striate cortex of Long-Evans hooded intact, monocular visual deprivation (MD) and MD treated with NGF rats. The averaged visual evoked responses (AVER) were obtained from both hemispheres and provided comparison after binocular photic stimuli between the contralateral and the ipsilateral striate cortex with relation to the MD eye. One month of monocular visual deprivation at the critical period of development resulted in marked reduction of the amplitudes of AVER components as compared to the control recordings (P0.001). These changes of the AVER could be prevented by NGF infusion to lateral ventricle at the dosage of 2.0-2.4 micrograms/day for four weeks during the monocular deprivation. In conclusion, the change of AVER amplitudes induced by monocular visual deprivation during the critical period of development can be prevented by NGF infusion to lateral ventricle.

Tyrosine hydroxylase (TH) is the key enzyme for synthesizing dopamine (DA) in dopaminergic neurons and its terminals. Emerging experimental and clinical evidence support the hypothesis of a disturbance in dopamine neurotransmission following traumatic brain injury (TBI). However, the effect of controlled cortical impact (CCI) injury on TH in the nigrostriatal system is currently unknown. To determine if there is an alteration in TH after CCI injury, we examined TH levels at 1 day, 7 days, and 28 days post-injury by utilizing a commercially available antibody specific to TH. Rats were anesthetized and surgically prepared for CCI injury (4 m/s, 3.2 mm) or sham surgery. Injured (N=6) and sham animals (N=6) were sacrificed and coronally sectioned (35 microm thick) through the striatum and substantia nigra (SN) for immunohistochemistry. Additionally, semiquantitative measurements of TH protein in striatal and SN homogenates from injured (N=6) and sham (N=6) rats sacrificed at the appropriate time post-surgery were assessed using Western blot analysis. TH protein is bilaterally increased at 28 days post-injury in nigrostriatal system revealed by immunohistochemistry in injured rats compared to sham controls. Western blot analysis confirms the findings of immunohistochemistry in both striatum and SN. We speculate that the increase in TH in the nigrostriatal system may reflect a compensatory response of dopaminergic neurons to upregulate their synthesizing capacity and a delayed increase in the efficiency of DA neurotransmission after TBI.

Disturbances in dopamine neurotransmission contribute to frontal lobe dysfunction after traumatic brain injury. The changes in dopamine neurotransmission may be mediated by alterations in the dopamine transporter, which plays a key role in maintaining dopamine homeostasis. To determine whether the dopamine transporter system is altered after traumatic brain injury, dopamine transporter protein was examined bilaterally in the rat frontal cortex by Western blot at I, 7, and 28 days after controlled cortical impact or sham injury (n = 6/group). Dopamine transporter protein expression was decreased in the injured (ipsilateral) cortex at 7 days and bilaterally at 28 days in injured vs sham control rats. The decrease in dopamine transporter protein levels may reflect a traumatic brain-injury-induced down-regulation of dopamine transporter and/or a loss of dopaminergic fibers.

Both the exogenous administration of fibroblast growth factor-2 (FGF-2) or the induction of moderate hypothermia have been shown to attenuate histopathology and improve functional outcome after traumatic brain injury (TBI). Since combined therapeutic strategies may be more beneficial than single therapies, we examined the potential synergistic effect of FGF-2 combined with moderate hypothermia treatment induced 10 min after TBI on functional and histological outcome following controlled cortical impact (CCI) injury. Fifty male Sprague-Dawley rats were randomized to one sham and four CCI treatment groups: Sham+vehicle (VEH); FGF-2 (45 microg/kg/h for 3 h i.v.)+Normothermia (37+/-0.5 degrees C); FGF-2+Hypothermia (32+/-0.5 degrees C for 3 h); VEH+Norm; VEH+Hypo. Vestibulomotor performance on the beam balance and beam-walk (BW) tasks on post-operative days 1-5 and spatial memory acquisition in the Morris water maze (MWM) on days 14-18 were assessed. After 4 weeks survival, histological evaluations (CA(1) and CA(3) cell counts and lesion volume) were performed. MWM performance improved in all treatment groups, but combined treatment was not more efficacious than either alone. The FGF-2+Hypo group performed significantly better than the other injured treatment groups in the BW task. Lastly, no significant group differences in beam balance or histological outcome were observed. These data suggest a suboptimal and incomplete synergy of combined FGF-2 and hypothermia treatment. These data may indicate that either our dose of FGF-2 or combination therapy was not optimized in our model.

Persistent cognitive deficits are one of the most important sequelae of head injury in humans. In an effort to model some of the structural and neuropharmacological changes that occur in chronic postinjury brains, we examined the longitudinal effects of moderate vertical controlled cortical impact (CCI) on place learning and memory using the Morris water maze (MWM) test, morphology, and vesicular acetylcholine (ACh) transporter (VAChT) and muscarinic receptor subtype 2 (M2) immunohistochemistry. Vertical CCI (left parietal cortex, 4 m/sec, 2.5 mm; n = 10) or craniotomy (sham) was produced in male Sprague-Dawley rats (n = 10). Place learning was tested at 2 weeks, 4 weeks, 3 months, 6 months, and 12 months postinjury with the escape platform in a different maze quadrant for each time point. At each interval, rats received 5 days of water maze acquisition (latency to find hidden platform), a probe trial to measure place memory, and 2 days of visible platform trials to control for nonspecific deficits. At 3 weeks, half the animals were sacrificed for histology. At these injury parameters, CCI produced no significant differences in place learning between injured and sham rats at 2 weeks, 4 weeks, or 6 months after injury. However, at 3 and 12 months, the injured rats took significantly longer to find the hidden platform than the sham rats. Probe trial performance differed only at 12 months postinjury between injured (25.73+/-2.1%, standard error of the mean) and sham rats (44.09+/-7.0%, p < 0.05). The maze deficits at 1 year were not due to a worsening of performance, but may have resulted from a reduced ability of injured rats to benefit from previous water maze experience. Hemispheric loss of 30.4+/-5.5 mm3 was seen at 3 weeks after injury (versus respective sham). However, hemispheric loss almost doubled by 1 year after injury (51.5+/-8.5 mm3, p < 0.05 versus all other groups). Progressive tissue loss was also reflected by a three- to fourfold increase in ipsilateral ventricular volume between 3 weeks and 1 year after injury. At 1 year after injury, immunostaining for VAChT was dramatically increased in all sectors of the hippocampus and cortex after injury. Muscarinic receptor subtype 2 (M2) immunoreactivity was dramatically decreased in the ipsilateral hippocampus. This suggests a compensatory response of cholinergic neurons to increase the efficiency of ACh neurotransmission. Moderate CCI in rats produces subtle MWM performance deficits accompanied by persistent alteration in M2 and VAChT immunohistochemistry and progressive tissue atrophy. The inability of injured rats to benefit from repeated exposures to the MWM may represent a deficit in procedural memory that is independent of changes in hippocampal cholinergic systems.

The expression of interleukin (IL)-1 beta, IL-6 and their respective receptors has been studied in the rat brain before and up to 24 h after injury. Messenger RNA transcripts of these four genes were detected by in situ hybridization (ISH) in different structures of the intact brain. The distribution was very similar for IL-1 beta, IL-6 and IL-6 receptor (IL-6R). The expression of IL-1R was more widespread. Within hours after injury, an increased expression of IL-1 beta, and thereafter of IL-6 was documented. The expression of IL-1R and IL-6R was also increased. This expression was bilateral and not restricted to the injured area. Within 24 h, all ISH patterns had returned to normal. The molecular data were confirmed by protein data. Indeed, the distribution of IL-6 (detected by immunocytochemistry) agreed with the ISH patterns for IL-6. Furthermore, extracellular fluid was collected by microdialysis at the site of the lesion during 12 h and successive fractions were assayed for the presence of bioactive IL-1 and IL-6. Increases in IL-1 and later in IL-6 levels were detected. The rapid and concomitant increased expression of IL-1 beta, IL-6 and their receptors after injury stresses their possible early role in inflammatory mechanisms also in the brain, before any recruitment of inflammatory cells from remote nervous and not nervous areas.

Leptin receptors (LR) mediate central (satiety) and peripheral metabolic actions of the translated and secreted product of the obese (ob) gene. While two isoforms as products of RNA splicing have been characterized, both of them bind leptin on the same epitope. Even though LR has been reported in adult tissues, limited information exists with respect to its presence and tissue-distribution in the embryo. We examined the spatial and temporal distribution of LR in the rat conceptus and placenta from d9 to d21 at 2 day intervals (term≈d21), and postnatal d15, and d30 (n=7/age/group). Employing frozen saggital embryonic and postnatal rat sections, 125I-leptin binding in the presence and absence of 0.6 × 10-6M unlabeled leptin was assessed by autoradiography and microdensitometry (Molecular Analyst Microdensitometry Program - 1.4.1, NIH). On d9, the placenta revealed LR while the known control (125I-insulin binding [IR]) was noted in amniotic membranes. At d11, LR was noted in the liver, while liver IR was present at d13. LR was present in the brain [d13], spine and long bones [d13], heart [d15], and kidney [d17]. The LR increased 2.5 fold in density from d9 to d21 (p < 0.01), with a subsequent 3-fold rise postnatally reaching a peak at d15 (p < 0.05). In contrast, IR demonstrated a 1.5-fold increase prenatally followed by a peak at postnatal d15. We conclude that 1] LR are present in embryonic tissues although the ontogenic peak is observed postnatally at d15, 2] LR are distributed in various tissues, with the earliest appearance being limited to the placenta and liver even before its appearance in the brain. We speculate that LR mediate the biological effects of leptin both centrally and peripherally in the embryo thereby regulating metabolism and growth.

Prenatal glucocorticoid (GC) therapy is an accepted practice in the prevention of respiratory distress syndrome in the neonate. Concerns regarding its use in early gestation and its side effects upon fetal and postnatal growth and metabolism persist. The recent cloning of the obese gene and obese gene receptors (OB-R) has shed some light upon mechanisms by which altered metabolism (anorexic/catabolic) and diminution in growth occurs. To determine if the ob gene (leptin) receptors mediate the growth reducing effects of GC, we intraperitoneally administered 2 doses of 0.3 mg/kg/day dexamethasone (GC) or vehicle (V) beginning d7, d9, d11, d13, d15, d17, and d19 (term ≈21d; n=7/age/group), and examined the binding of125 I-leptin (10-9M), in the presence and absence of 0.6 X 10-6M unlabeled leptin, to the d9, d11, d13, d15, d17, d19, and d21 frozen saggital embryonic liver sections. In addition, postnatal livers were studied on d15 and d30 (n=7/age/group) subsequent to GC or V administration on d19 gestation. Specific 125I-leptin binding was assessed by quantitation of the autoradiographs and phoshoimages by microdensitometry(Molecular Analyst Microdensitometry Program-1.4.1, NIH). GC reduced body weight at all ages (p < 0.001), and increased hepatic 125I-leptin binding at all ages with a maximal 3-fold increase noted at postnatal d15 (p< 0.005). This is in contrast to a 2-fold decline in hepatic125 I-insulin binding (p < 0.01) at postnatal d15, which served as a known control. We conclude that prenatal GC 1] increases hepatic leptin receptors from mid- to late gestation, 2] has a persistent enhancing effect upon postnatal hepatic leptin receptors. We speculate that prenatal GC increases the biological action of leptin peripherally, thereby affecting the metabolic mileu and the growth potential of the fetus and the newborn.