Dorothy A. Kozlowski

Use-dependent exacerbation of brain damage occurs during an early post-lesion vulnerable period.

For a period of time after unilateral brain injury, surviving neural tissue surrounding the lesion may be vulnerable to extremely high behavioral demand. Previously, we found that lesions of the forelimb representation area of the sensorimotor cortex (FL-SMC) in rats increase in size substantially when the intact forelimb is immobilized with a plaster of paris cast during the first 15 days after surgery, which forces overuse of the impaired forelimb. The present study was designed to determine whether the adult brain is more vulnerable to forced overuse of the impaired forelimb during the first 7 days post-lesion than during the second 7 days post-lesion. Using behavioral tests of forelimb use and stereological analysis of remaining tissue volume 40 days after FL-SMC lesions, we found that forced overuse of the impaired forelimb during the first 7 days after the initial damage caused expansion of neural injury and greatly interfered with restoration of function. In contrast, forced overuse of the impaired forelimb during the second 7 days had no significant effect on lesion size but nevertheless interfered with restoration of function. Thus, surviving neural tissue in the damaged hemisphere and recovery of function appear to be vulnerable to prolonged forced overuse of the impaired forelimb throughout the first 15 days, but tissue loss was detectable only when the animal was forced to use the impaired forelimb during the first 7 days after injury.

Differential effects of glial cell line-derived neurotrophic factor (GDNF) in the striatum and substantia nigra of the aged Parkinsonian rat

Injection of an adenoviral (Ad) vector encoding human glial cell line-derived neurotrophic factor (GDNF) protects dopaminergic (DA) neurons in the substantia nigra (SN) of young rats. As Parkinson’s disease occurs primarily in aged populations, we examined whether chronic biosynthesis of GDNF, achieved by adenovirus-mediated delivery of a GDNF gene (AdGDNF), can protect DA neurons and improve DA-dependent behavioral function in aged (20 months) rats with progressive 6-OHDA lesions of the nigrostriatal projection. Furthermore, the differential effects of injecting AdGDNF either near DA cell bodies in the SN or at DA terminals in the striatum were compared. AdGDNF or control vector was injected unilaterally into either the striatum or SN. One week later, rats received a unilateral intrastriatal injection of 6-OHDA on the same side as the vector injection. AdGDNF injection into either the striatum or SN significantly reduced the loss of FG labelled DA neurons 5 weeks after lesion (P ⩽ 0.05). However, only striatal injections of AdGDNF protected against the development of behavioral deficits characteristic of unilateral DA depletion. Striatal AdGDNF injections also reduced tyrosine hydroxylase fiber loss and increased amphetamine-induced striatal Fos expression. These results demonstrate that increased levels of striatal, but not nigral, GDNF biosynthesis prevents DA neuronal loss and protects DA terminals from 6-OHDA-induced damage, thereby maintaining DA function in the aged rat.

Cellular proliferation and migration following a controlled cortical impact in the mouse.

Neurogenesis following neural degeneration has been demonstrated in many models of disease and injury. The present study further examines the early proliferative and migratory response of the brain to a controlled cortical impact (CCI) model of traumatic brain injury. The CCI was centered over the forelimb sensorimotor cortex, unilaterally, in the adult mouse. To examine proliferation, bromo-deoxyuridine (BrdU) was injected i.p. immediately post-injury and on post-injury days 1, 2, and 3. To assess migration, we labeled SVZ cells with inert latex microspheres immediately post-injury. By combining microsphere labeling with BrdU, we determined if migrating cells had gone through the S-phase of the cell cycle after the lesion. In addition, we used a marker of neurogenesis and migration, doublecortin, to further characterize the response of the SVZ to the injury. Lastly, we determined whether subregions of the SVZ respond differentially to injury. The current study demonstrates that 3 days following CCI cellular proliferation is seen around the cortex, in the SVZ, corpus callosum, and subcortical areas anatomically connected to, but not directly damaged by the impact. It delineates that an increase in proliferation occurs in the dorsal-most aspect of the ipsilateral SVZ following impact. Lastly, it demonstrates that proliferating cells migrate from the SVZ to cortical and subcortical structures affected by the injury and that some of these cells are migrating neuroblasts.

Delivery of a GDNF Gene into the Substantia Nigra after a Progressive 6-OHDA Lesion Maintains Functional Nigrostriatal Connections

The effects of delivering GDNF via an adenoviral vector (AdGDNF) 1 week after lesioning dopaminergic neurons in the rat substantia nigra (SN) with 6-hydroxydopamine (6-OHDA) were examined. Rats were unilaterally lesioned by injection of 6-OHDA into the striatum, resulting in progressive degeneration of dopaminergic neurons in the SN. One week later, when substantial damage had already occurred, AdGDNF or a control vector harboring beta-galactosidase (AdLacZ) was injected into either the striatum or SN (3.2 x 10(7) PFU/microl in 2 microl). Rats were examined behaviorally with the amphetamine-induced rotation test and for forelimb use for weight-bearing movements. On day 30 postlesion, the extent of nigrostriatal tract degeneration was determined by injecting a retrograde tracer (FluoroGold) bilaterally into the lesioned striatum. Five days later, rats were sacrificed within 2 h of amphetamine injection to examine amphetamine-induced Fos expression in the striatum, a measure of dopaminergic-dependent function in target neurons. AdGDNF injection in the SN rescued dopaminergic neurons in the SN and increased the number of dopaminergic neurons that maintained a connection to the striatum, compared to rats injected with AdLacZ. Further support that these spared SN cells maintained functional connections to the striatum was evidenced by increased Fos expression in striatal target neurons and a decrease in amphetamine-induced rotation. In contrast to the effects observed in rats injected with AdGDNF in the SN, rats injected with AdGDNF in the striatum did not exhibit significant ameliorative effects. This study demonstrates that experimentally increasing levels of GDNF biosynthesis near the dopaminergic neuronal soma is effective in protecting the survival of these neurons and their function even when therapy is begun after 6-OHDA-induced degeneration has commenced. Thus, GDNF gene therapy may ameliorate the consequences of Parkinsons disease through rescuing compromised dopaminergic neurons.

Use-Dependent Exaggeration of Brain Injury: Is Glutamate Involved?

Extreme overreliance on the impaired forelimb following unilateral lesions of the forelimb representation area of the rat sensorimotor cortex (FL-SMC) leads to exaggeration of the initial cortical injury. Glutamate has repeatedly been implicated in the secondary processes leading to neuronal death following traumatic insult, chiefly because of the neuroprotective properties of excitatory amino acid antagonists in a variety of animal models of brain injury. The present study investigated the possibility that NMDA receptor-mediated processes are involved in use-dependent exaggeration of neuronal injury. Rats were fitted with one-sleeved casts that immobilized the intact forelimb for the first 7 days following FL-SMC lesion, a procedure previously shown to result in use-dependent exaggeration of injury and more severe and persistent limb-use deficits. In the present investigation, administration of MK-801 (1 mg/kg ip once daily on alternate days) during the casting period spared neural tissue surrounding the lesion and enhanced functional recovery of the impaired forelimb. These results suggest a role for NMDA receptor-mediated processes in use-dependent exaggeration of injury.

Differential activation of microglia in neurogenic versus non-neurogenic regions of the forebrain.

Proliferation decreases in the neurogenic subventricular zone (SVZ) of mice after aspiration lesions of the cerebral cortex. We hypothesized that microglial activation may contribute to this given microglial activation attenuates neurogenesis in the hippocampus. Using CD45, CD11b, IB4, and IL‐6 immunohistochemistry (IHC), BrdU IHC, and fluorescent bead tracking of peripheral monocytes into the brain, we compared microglial activation in the SVZ to non‐neurogenic forebrain regions. SVZ microglia exhibited greater constitutive activation and proliferation than did microglia in non‐neurogenic regions. In contrast to the SVZ, the dentate gyrus (DG) contained relatively few CD45+ cells. After aspiration cerebral cortex lesions, microglia became activated in the cerebral cortex, corpus callosum, and striatum. SVZ microglial activation did not increase, and similarly, microglia in the DG were less activated after injury than in adjacent non‐neurogenic regions. We next showed that SVZ microglia are not categorically refractory to activation, since deep cortical contusion injuries increased SVZ microglial activation. Macrophages migrate into the brain during development, but it is unclear if this is recapitulated after injury. Infiltration of microbead‐labeled macrophages into the brain did not change after injury, but resident SVZ microglia were induced to migrate toward the injury. Our data show that both constitutive and postlesion levels of microglial activation differ between neurogenic and non‐neurogenic regions.

Glial cell line-derived neurotrophic factor (GDNF) gene delivery protects dopaminergic terminals from degeneration.

Previously, we observed that injection of an adenoviral (Ad) vector expressing glial cell line-derived neurotrophic factor (GDNF) into the striatum, but not the substantia nigra (SN), prior to a partial 6-OHDA lesion protects dopaminergic (DA) neuronal function and prevents the development of behavioral impairment in the aged rat. This suggests that striatal injection of AdGDNF maintains nigrostriatal function either by protecting DA terminals or by stimulating axonal sprouting to the denervated striatum. To distinguish between these possible mechanisms, the present study examines the effect of GDNF gene delivery on molecular markers of DA terminals and neuronal sprouting in the aged (20 month) rat brain. AdGDNF or a control vector coding for beta-galactosidase (AdLacZ) was injected unilaterally into either the striatum or the SN. One week later, rats received a unilateral intrastriatal injection of 6-OHDA on the side of vector injection. Two weeks postlesion, rats injected with AdGDNF into either the striatum or the SN exhibited a reduction in the area of striatal denervation and increased binding of the DA transporter ligand [(125)I]IPCIT in the lesioned striatum compared to control animals. Furthermore, injections of AdGDNF into the striatum, but not the SN, increased levels of tyrosine hydroxylase mRNA in lesioned DA neurons in the SN and prevented the development of amphetamine-induced rotational asymmetry. In contrast, the level of T1 alpha-tubulin mRNA, a marker of neuronal sprouting, was not increased in lesioned DA neurons in the SN following injection of AdGDNF either into the striatum or into the SN. These results suggest that GDNF gene delivery prior to a partial lesion ameliorates damage caused by 6-OHDA in aged rats by inhibiting the degeneration of DA terminals rather than by inducing sprouting of nigrostriatal axons.

Adenovirus-Mediated Transgene Expression in Nonhuman Primate Brain

Transgene expression in the brain of St. Kitts green monkey, Cercopithecus aethiops sabeus, was studied following injection of a serotype 5 adenoviral vector deleted in E1 and E3. The vector harbored the transgene for Escherichia coli beta-galactosidase (beta-Gal) with the simian virus 40 (SV40) nuclear localization signal under control of the Rous sarcoma viral (RSV) long terminal repeat. Several titers ranging from 5 x 10(7) to 2 x 10(9) plaque-forming units (PFU) in volumes ranging from 5 to 250 microl were injected into the caudate nuclei of 18 monkeys. Monkeys were treated with dexamethasone for 9 days, beginning the day prior to surgery, and were sacrificed at 1 week or at 1, 2, or 3 months. At 1 week, beta-Gal was expressed in thousands of cells, including both neurons and astrocytes. In addition, some dopaminergic neurons in the substantia nigra expressed transgene, suggesting retrograde transport of the vector. At 1 month 162,000+/-68,000 (SEM) or 65,000+/-29,000 beta-Gal-expressing cells persisted in striatum injected with 6 x 10(8) PFU in 30 microl or 5 x 10(7) PFU in 5 microl, respectively. Transgene expression was also observed in one of two monkeys sacrificed at 2 months and in a single monkey sacrificed at 3 months. No transgene expression was observed at 1 month in striatum injected with a higher titer (2 x 10(9) PFU in 100 microl) or more dilute vector (5 x 10(7) PFU in 30 microl). Staining for the major histocompatibility complex II (MHC II) subtype DR showed intense staining in sites injected with a higher vector titer, in which no transgene persisted at 1 month, whereas low to moderate staining was present in sites with high transgene expression. These observations suggest that there is an optimal range of vector titers for obtaining persistent transgene expression from E1E3-deleted adenovirus in primate brain, above which host responses limit transgene stability.

Relationship between dendritic pruning and behavioral recovery following sensorimotor cortex lesions

A unilateral injury to the forelimb area of the sensorimotor cortex results in an increase in dendritic arborization in the contralateral homotopic cortex which is followed by a pruning back of these dendritic arbors. The increase in arborization is due to an increase in the use of the unimpaired forelimb for postural-motor support; whereas, the dendritic pruning is related, in time, to the return to more symmetrical limb use, but is not prevented by the maintenance of asymmetrical limb use. Dendritic pruning can be prevented by administering an NMDA receptor antagonist (such as MK801 or ethanol) during the pruning phase. This manipulation also coincides with the chronic reinstatement of behavioral deficits. The purpose of this study was to see whether removing the antagonism of the NMDA receptor results in the eventual return of dendritic pruning and behavioral recovery. Therefore, MK801 was administered to lesioned animals starting at post-lesion day 18. One group received MK801 injections until day 60 (Lesion + MK60) and another lesioned group received MK801 until day 30 after which the injections were changed to saline until day 60 (Lesion + MK30). Lesion + MK60 animals showed a prevention of dendritic pruning as well as a chronic reinstatement of forelimb deficits. Lesion + MK30 animals also showed a prevention of dendritic pruning, however, they showed behavioral recovery. These findings suggest that pruning of dendritic arbors may not be directly related to behavioral recovery.

Targeted gene inactivation of calpain-1 suppresses cortical degeneration due to traumatic brain injury and neuronal apoptosis induced by oxidative stress.

Background: Calpains play an important role in the regulation of cell death. Results: Calpain-1 inhibition decreases cortical neurodegeneration following TBI by regulating calcium influx and apoptosis of neurons under oxidative stress. Conclusion: Genetic inhibition of calpain-1 reduces neurodegeneration and suppresses neuronal apoptosis. Significance: Targeted inhibition of calpain-1 offers a promising therapeutic approach against TBI and other neurodegenerative diseases. Calpains are calcium-regulated cysteine proteases that have been implicated in the regulation of cell death pathways. Here, we used our calpain-1 null mouse model to evaluate the function of calpain-1 in neural degeneration following a rodent model of traumatic brain injury. In vivo, calpain-1 null mice show significantly less neural degeneration and apoptosis and a smaller contusion 3 days post-injury than wild type littermates. Protection from traumatic brain injury corroborated with the resistance of calpain-1 neurons to apoptosis induced by oxidative stress. Biochemical analysis revealed that caspase-3 activation, extracellular calcium entry, mitochondrial membrane permeability, and release of apoptosis-inducing factor from mitochondria are partially blocked in the calpain-1 null neurons. These findings suggest that the calpain-1 knock-out mice may serve as a useful model system for neuronal protection and apoptosis in traumatic brain injury and other neurodegenerative disorders in which oxidative stress plays a role.