Many mechanisms or pathways are involved in secondary post-traumatic brain injury, such as the ub... more Many mechanisms or pathways are involved in secondary post-traumatic brain injury, such as the ubiquitin-proteasome pathway (UPP), axonal degeneration and neuronal cell apoptosis. UCH-L1 is a protein that is expressed in high levels in neurons and may have important roles in the UPP, autophagy and axonal integrity. The current study aims to evaluate the role of UCH-L1 in post-traumatic brain injury (TBI) and its potential therapeutic effects. A novel protein was constructed that fused the protein transduction domain (PTD) of trans-activating transduction (TAT) protein with UCH-L1 (TAT-UCH-L1) in order to promote neuronal transduction. The TAT-UCH-L1 protein was readily detected in brain by immunoblotting and immunohistochemistry after i.p. administration in mice. TBI was induced in mice using the controlled cortical impact (CCI) model. TAT-UCH-L1 treatment significantly attenuated K48-linkage polyubiquitin (polyUb)-protein accumulation in hippocampus after CCI compared to vehicle co...
Cyclooxygenase, or prostaglandin G/H synthase, is the rate-limiting step in the production of pro... more Cyclooxygenase, or prostaglandin G/H synthase, is the rate-limiting step in the production of prostaglandins. A new isoform, cyclooxygenase-2 (COX-2), has been cloned that is induced during inflammation in leukocytes and by synaptic activity in neurons. The objectives of this study are to determine the nature of COX-2 expression in normal and traumatized rat spinal cord, and to determine the effects of selective COX-2 inhibition on functional recovery following spinal cord injury. Using a weight-drop model of spinal cord injury, COX-2 mRNA expression was studied with in situ hybridization. COX-2 protein expression was examined by immunohistochemistry and Western analysis. Finally, using the highly selective COX-2 inhibitor, 1-[(4-methylsufonyl)phenyl]-3-tri-fluro-methyl-5-[(4-flur o)phenyl]prazole (SC58125), the effect of COX-2 inhibition on functional outcome following a spinal cord injury was determined. COX-2 was expressed in the normal adult rat spinal cord. COX-2 mRNA and protein production were increased following injury with increases in COX-2 mRNA production detectable at 2 h following injury. Increased levels of COX-2 protein were detectable for at least 48 h following traumatic spinal cord injury. Selective inhibition of COX-2 activity with SC58125 resulted in improved mean Basso, Beattie, and Bresnahan scores in animals with 12.5- and 25-g/cm spinal cord injuries; however, the effect was significant only for the 12.5g/cm injury group (p=0.0001 vs. p=0.0643 in the 25-g/cm group). These data demonstrate that COX-2 mRNA and protein expression are induced by spinal cord injury, and that selective inhibition of COX-2 improves functional outcome following experimental spinal cord injury.
Expression of the proto-oncogene bcl-2 prevents programmed cell death in vitro, but it is not kno... more Expression of the proto-oncogene bcl-2 prevents programmed cell death in vitro, but it is not known whether bcl-2 plays a role in determining cell survival after cerebral ischemia. Using immunohistochemistry and Western blot analysis, bcl-2 protein expression was studied in the rat brain 24 h following 60 or 120 min of temporary focal ischemia. Sixty minutes of ischemia induced bcl-2 protein in neurons throughout the frontoparietal cortex in non-infarcted regions, whereas 120 min of ischemia induced bcl-2 in neurons only just outside the margin of the infarction. bcl-2 protein was also induced in glial cells, mainly microglia, border zone of the infarction. In the infarcted regions of caudate and cortex, bcl-2 protein was exclusively induced in endothelial cells and the vessel walls. Western blot revealed a characteristic single band at 26 kDa only in ischemic samples. These data show that bcl-2 is induced in sublethally injured cells and suggest that bcl-2 could play a role in determining cell survival in cerebral ischemia.
The rheoencephalogram (REG) is the change in the electrical impedance of the head that occurs wit... more The rheoencephalogram (REG) is the change in the electrical impedance of the head that occurs with each heart beat. Without knowledge of the relationship between cerebral blood flow (Q) and the REG, the utility of the REG in the study of the cerebral vasculature is greatly limited. The hypothesis is that the relationship between the REG and Q when venous outflow is nonpulsatile is^ (DIAGRAM, TABLE OR GRAPHIC OMITTED...PLEASE SEE DAI)^ where K is a proportionality constant and Q is the mean Q.^ Pulsatile CBF was measured in the goat via a chronically implanted electromagnetic flowmeter. Electrodes were implanted in the ipsilateral cerebral hemisphere, and the REG was measured with a two electrode impedance plethysmograph. Measurements were made with the animal's head elevated so that venous flow pulsations were not transmitted from the heart to the cerebral veins. Measurements were made under conditions of varied cerebrovascular resistance induced by altering blood CO(,2) levels and under conditions of high and low cerebrospinal fluid pressures. There was a high correlation (r = .922-.983) between the REG calculated from the hypothesized relationship and the measured REG under all conditions.^ Other investigators have proposed that the REG results from linear changes in blood resistivity proportional to blood velocity. There was little to no correlation between the measured REG and the flow velocity ( r = .022-.306). A linear combination of the flow velocity and the hypothesized relationship between the REG and Q did not predict the measured REG significantly better than the hypothesized relationship alone in 37 out of 50 experiments.^ Jacquy proposed an index (F) of cerebral blood flow calculated from amplitudes and latencies of the REG. The F index was highly correlated (r = .929) with measured cerebral blood flow under control and hypercapnic conditions, but was not as highly correlated under conditions of hypocapnia (r = .723) and arterial hypotension (r = .681).^ The results demonstrate that the REG is not determined by mean cerebral blood flow, but by the pulsatile flow only. Thus, the utility of the REG in the determination of mean cerebral blood flow is limited. ^
Gene expression plays an important role in determining the fate of neurons after ischemia. To ide... more Gene expression plays an important role in determining the fate of neurons after ischemia. To identify additional genes that promote survival or execute programmed cell death in ischemic neurons, a subtractive cDNA library was constructed from hippocampus of rats subjected to global ischemia. With use of a differential screening technique, a cDNA was identified that was up-regulated after ischemia. The cDNA was found to have high homology with human cyclin H at both the nucleotide level (89%) and the amino acid level (93%). Northern blotting detected cyclin H mRNA in nonischemic and ischemic brains. In situ hybridization studies revealed that cyclin H message was found in hippocampal neurons in nonischemic brain. After ischemia, expression was increased primarily in the dentate gyrus and CA3 regions of hippocampus. Expression of cyclin H protein, detected by western blotting of hippocampal tissue, was increased after global ischemia, but expression of cyclins B1 and D1 and other related cell cycle genes (Cdk7 and Cdc2) was not increased. Cyclin H immunoreactivity was found exclusively within neurons. After ischemia, there was increased immunoreactivity within neurons in dentate gyrus, CA3, and cortex. Thus, cyclin H is expressed in normal postmitotic neurons and expression is increased in neurons that are ischemic yet survive. These results suggest that cyclin H may have functions in neurons other than cell cycle regulation, including other known functions such as DNA repair.
During programmed cell death, activation of caspase-3 leads to proteolysis of DNA repair proteins... more During programmed cell death, activation of caspase-3 leads to proteolysis of DNA repair proteins, cytoskeletal proteins, and the inhibitor of caspase-activated deoxyribonuclease, culminating in morphologic changes and DNA damage defining apoptosis. The participation of caspase-3 activation in the evolution of neuronal death after traumatic brain injury in rats was examined. Cleavage of pro-caspase-3 in cytosolic cellular fractions and an increase in caspase-3-like enzyme activity were seen in injured brain versus control. Cleavage of the caspase-3 substrates DNA-dependent protein kinase and inhibitor of caspase-activated deoxyribonuclease and co-localization of cytosolic caspase-3 in neurons with evidence of DNA fragmentation were also identified. Intracerebral administration of the caspase-3 inhibitor N-benzyloxycarbonyl-Asp-Glu-Val-Asp-fluoromethyl ketone (480 ng) after trauma reduced caspase-3-like activity and DNA fragmentation in injured brain versus vehicle at 24 h. Treatment with N-benzyloxycarbonyl-Asp-Glu-Val-Asp-fluoromethyl ketone for 72 h (480 ng/day) reduced contusion size and ipsilateral dorsal hippocampal tissue loss at 3 weeks but had no effect on functional outcome versus vehicle. These data demonstrate that caspase-3 activation contributes to brain tissue loss and downstream biochemical events that execute programmed cell death after traumatic brain injury. Caspase inhibition may prove efficacious in the treatment of certain types of brain injury where programmed cell death occurs.
Many mechanisms or pathways are involved in secondary post-traumatic brain injury, such as the ub... more Many mechanisms or pathways are involved in secondary post-traumatic brain injury, such as the ubiquitin-proteasome pathway (UPP), axonal degeneration and neuronal cell apoptosis. UCH-L1 is a protein that is expressed in high levels in neurons and may have important roles in the UPP, autophagy and axonal integrity. The current study aims to evaluate the role of UCH-L1 in post-traumatic brain injury (TBI) and its potential therapeutic effects. A novel protein was constructed that fused the protein transduction domain (PTD) of trans-activating transduction (TAT) protein with UCH-L1 (TAT-UCH-L1) in order to promote neuronal transduction. The TAT-UCH-L1 protein was readily detected in brain by immunoblotting and immunohistochemistry after i.p. administration in mice. TBI was induced in mice using the controlled cortical impact (CCI) model. TAT-UCH-L1 treatment significantly attenuated K48-linkage polyubiquitin (polyUb)-protein accumulation in hippocampus after CCI compared to vehicle co...
Cyclooxygenase, or prostaglandin G/H synthase, is the rate-limiting step in the production of pro... more Cyclooxygenase, or prostaglandin G/H synthase, is the rate-limiting step in the production of prostaglandins. A new isoform, cyclooxygenase-2 (COX-2), has been cloned that is induced during inflammation in leukocytes and by synaptic activity in neurons. The objectives of this study are to determine the nature of COX-2 expression in normal and traumatized rat spinal cord, and to determine the effects of selective COX-2 inhibition on functional recovery following spinal cord injury. Using a weight-drop model of spinal cord injury, COX-2 mRNA expression was studied with in situ hybridization. COX-2 protein expression was examined by immunohistochemistry and Western analysis. Finally, using the highly selective COX-2 inhibitor, 1-[(4-methylsufonyl)phenyl]-3-tri-fluro-methyl-5-[(4-flur o)phenyl]prazole (SC58125), the effect of COX-2 inhibition on functional outcome following a spinal cord injury was determined. COX-2 was expressed in the normal adult rat spinal cord. COX-2 mRNA and protein production were increased following injury with increases in COX-2 mRNA production detectable at 2 h following injury. Increased levels of COX-2 protein were detectable for at least 48 h following traumatic spinal cord injury. Selective inhibition of COX-2 activity with SC58125 resulted in improved mean Basso, Beattie, and Bresnahan scores in animals with 12.5- and 25-g/cm spinal cord injuries; however, the effect was significant only for the 12.5g/cm injury group (p=0.0001 vs. p=0.0643 in the 25-g/cm group). These data demonstrate that COX-2 mRNA and protein expression are induced by spinal cord injury, and that selective inhibition of COX-2 improves functional outcome following experimental spinal cord injury.
Expression of the proto-oncogene bcl-2 prevents programmed cell death in vitro, but it is not kno... more Expression of the proto-oncogene bcl-2 prevents programmed cell death in vitro, but it is not known whether bcl-2 plays a role in determining cell survival after cerebral ischemia. Using immunohistochemistry and Western blot analysis, bcl-2 protein expression was studied in the rat brain 24 h following 60 or 120 min of temporary focal ischemia. Sixty minutes of ischemia induced bcl-2 protein in neurons throughout the frontoparietal cortex in non-infarcted regions, whereas 120 min of ischemia induced bcl-2 in neurons only just outside the margin of the infarction. bcl-2 protein was also induced in glial cells, mainly microglia, border zone of the infarction. In the infarcted regions of caudate and cortex, bcl-2 protein was exclusively induced in endothelial cells and the vessel walls. Western blot revealed a characteristic single band at 26 kDa only in ischemic samples. These data show that bcl-2 is induced in sublethally injured cells and suggest that bcl-2 could play a role in determining cell survival in cerebral ischemia.
The rheoencephalogram (REG) is the change in the electrical impedance of the head that occurs wit... more The rheoencephalogram (REG) is the change in the electrical impedance of the head that occurs with each heart beat. Without knowledge of the relationship between cerebral blood flow (Q) and the REG, the utility of the REG in the study of the cerebral vasculature is greatly limited. The hypothesis is that the relationship between the REG and Q when venous outflow is nonpulsatile is^ (DIAGRAM, TABLE OR GRAPHIC OMITTED...PLEASE SEE DAI)^ where K is a proportionality constant and Q is the mean Q.^ Pulsatile CBF was measured in the goat via a chronically implanted electromagnetic flowmeter. Electrodes were implanted in the ipsilateral cerebral hemisphere, and the REG was measured with a two electrode impedance plethysmograph. Measurements were made with the animal's head elevated so that venous flow pulsations were not transmitted from the heart to the cerebral veins. Measurements were made under conditions of varied cerebrovascular resistance induced by altering blood CO(,2) levels and under conditions of high and low cerebrospinal fluid pressures. There was a high correlation (r = .922-.983) between the REG calculated from the hypothesized relationship and the measured REG under all conditions.^ Other investigators have proposed that the REG results from linear changes in blood resistivity proportional to blood velocity. There was little to no correlation between the measured REG and the flow velocity ( r = .022-.306). A linear combination of the flow velocity and the hypothesized relationship between the REG and Q did not predict the measured REG significantly better than the hypothesized relationship alone in 37 out of 50 experiments.^ Jacquy proposed an index (F) of cerebral blood flow calculated from amplitudes and latencies of the REG. The F index was highly correlated (r = .929) with measured cerebral blood flow under control and hypercapnic conditions, but was not as highly correlated under conditions of hypocapnia (r = .723) and arterial hypotension (r = .681).^ The results demonstrate that the REG is not determined by mean cerebral blood flow, but by the pulsatile flow only. Thus, the utility of the REG in the determination of mean cerebral blood flow is limited. ^
Gene expression plays an important role in determining the fate of neurons after ischemia. To ide... more Gene expression plays an important role in determining the fate of neurons after ischemia. To identify additional genes that promote survival or execute programmed cell death in ischemic neurons, a subtractive cDNA library was constructed from hippocampus of rats subjected to global ischemia. With use of a differential screening technique, a cDNA was identified that was up-regulated after ischemia. The cDNA was found to have high homology with human cyclin H at both the nucleotide level (89%) and the amino acid level (93%). Northern blotting detected cyclin H mRNA in nonischemic and ischemic brains. In situ hybridization studies revealed that cyclin H message was found in hippocampal neurons in nonischemic brain. After ischemia, expression was increased primarily in the dentate gyrus and CA3 regions of hippocampus. Expression of cyclin H protein, detected by western blotting of hippocampal tissue, was increased after global ischemia, but expression of cyclins B1 and D1 and other related cell cycle genes (Cdk7 and Cdc2) was not increased. Cyclin H immunoreactivity was found exclusively within neurons. After ischemia, there was increased immunoreactivity within neurons in dentate gyrus, CA3, and cortex. Thus, cyclin H is expressed in normal postmitotic neurons and expression is increased in neurons that are ischemic yet survive. These results suggest that cyclin H may have functions in neurons other than cell cycle regulation, including other known functions such as DNA repair.
During programmed cell death, activation of caspase-3 leads to proteolysis of DNA repair proteins... more During programmed cell death, activation of caspase-3 leads to proteolysis of DNA repair proteins, cytoskeletal proteins, and the inhibitor of caspase-activated deoxyribonuclease, culminating in morphologic changes and DNA damage defining apoptosis. The participation of caspase-3 activation in the evolution of neuronal death after traumatic brain injury in rats was examined. Cleavage of pro-caspase-3 in cytosolic cellular fractions and an increase in caspase-3-like enzyme activity were seen in injured brain versus control. Cleavage of the caspase-3 substrates DNA-dependent protein kinase and inhibitor of caspase-activated deoxyribonuclease and co-localization of cytosolic caspase-3 in neurons with evidence of DNA fragmentation were also identified. Intracerebral administration of the caspase-3 inhibitor N-benzyloxycarbonyl-Asp-Glu-Val-Asp-fluoromethyl ketone (480 ng) after trauma reduced caspase-3-like activity and DNA fragmentation in injured brain versus vehicle at 24 h. Treatment with N-benzyloxycarbonyl-Asp-Glu-Val-Asp-fluoromethyl ketone for 72 h (480 ng/day) reduced contusion size and ipsilateral dorsal hippocampal tissue loss at 3 weeks but had no effect on functional outcome versus vehicle. These data demonstrate that caspase-3 activation contributes to brain tissue loss and downstream biochemical events that execute programmed cell death after traumatic brain injury. Caspase inhibition may prove efficacious in the treatment of certain types of brain injury where programmed cell death occurs.
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