Probing Flow-Induced Biomolecular Interactions With Micro-Extensional Rheology: Tau Protein Aggregation.

Biomolecules in solutions subjected to extensional strain can form aggregates, which may be important for our understanding of pathologies involving insoluble protein structures where mechanical forces are thought to be causative (e.g., tau fibers in chronic traumatic encephalopathy (CTE)). To examine the behavior of biomolecules in solution under mechanical strains requires applying rheological methods, often to very small sample volumes. There were two primary objectives in this investigation: (1) To probe flow-induced aggregation of proteins in microliter-sized samples and (2) To test the hypothesis that tau protein aggregates under extensional flow. Tau protein (isoform:3R 0 N; 36.7 kDa) was divided into 10 µl droplets and subjected to extensional strain in a modified tensiometer. Sixteen independent tests were performed where one test on a single droplet comprised three extensional events. To assess the rheological performance of the fluid/tau mixture, the diameter of the filament that formed during extension was tracked as function of time and analyzed for signs of aggregation (i.e., increased relaxation time). The results were compared to two molecules of similar and greater size (Polyethylene Oxide: PEO35, 35 kDa and PEO100, 100 kDa). Analysis showed that the tau protein solution and PEO35 are likely to have formed aggregates, albeit at relatively high extensional strain rates (∼10 kHz). The investigation demonstrates an extensional rheological method capable of determining the properties of protein solutions in µl volumes and that tau protein can aggregate when exposed to a single extensional strain with potentially significant biological implications.

Mechanisms underlying vulnerabilities after repeat mild traumatic brain injuries.

Traumatic brain injury (TBI) has drawn national attention for its high incidence and mechanistic complexity. The majority of TBI cases are "mild" in nature including concussions and mild TBI (mTBI). Concussions are a distinct form of mTBI where diagnosis is difficult, quantification of the incidence is challenging and there is greater risk for subsequent injuries. While concussions occur in the general population, it has become a hallmark injury consistently observed among adolescent and young adult athletes and the risks for repeat TBI (rTBI) is significant. Clinical and experimental evidence shows that the magnitude and duration of deficits is dependent on the number and the interval between injuries. Several studies suggest that metabolic vulnerabilities after injury may contribute to the window for cerebral vulnerability from rTBI. In addition to metabolism, this review addresses how age, sex and hormones also play an important role in the response to repeat concussions. Understanding how these factors collectively contribute to concussion and rTBI recovery is critically important in establishing age/sex appropriate return to play guidelines, injury prevention, therapeutic interventions and mitigation of long-term consequences of rTBI.

Targeting the Epidemic: Interventions and Follow-up Are Necessary in the Pediatric Traumatic Brain Injury Clinic.

Traumatic brain injury is a major public health problem in the pediatric population. Previously, management was acute emergency department/primary care evaluation with follow-up by primary care. However, persistent symptoms after traumatic brain injury are common, and many do not have access to a specialized traumatic brain injury clinic to manage chronic issues. The goal of this study was to determine the factors related to outcomes, and identify the interventions provided in this subspecialty clinic. Data were extracted from medical records of 151 retrospective and 403 prospective patients. Relationships between sequelae, injury characteristics, and clinical interventions were analyzed. Most patients returning to clinic were not fully recovered from their injury. Headaches were more common after milder injuries, and seizures were more common after severe. The majority of patients received clinical intervention. The presence of persistent sequelae for traumatic brain injury patients can be evaluated and managed by a specialty concussion/traumatic brain injury clinic ensuring that medical needs are met.

Repeat traumatic brain injury in the developing brain.

The Center for Disease Control estimates that there are 1.7 million brain injuries in the US each year with 51% of these injuries occurring during periods of cerebral development. Among this population there is a growing population of individuals with repeat traumatic brain injury (RTBI). While the exact incidence is unknown, estimates range from 5.6 to 36% of the TBI population. This review summarizes the clinical problems/challenges and experimental research models that currently exist. It is intended to reveal the critical areas that need to be addressed so that age-relevant clinical management guidelines can be established to protect this population.

Repeat traumatic brain injury in the juvenile rat is associated with increased axonal injury and cognitive impairments.

Among the enormous population of head-injured children and young adults are a growing subpopulation who experience repeat traumatic brain injury (RTBI). The most common cause of RTBI in this age group is sports-related concussions, and athletes who have experienced a head injury are at greater risk for subsequent TBI, with consequent long-term cognitive dysfunction. While several animal models have been proposed to study RTBI, they have been shown to either produce injuries too severe, were conducted in adults, involved craniotomy, or failed to show behavioral deficits. A closed head injury model for postnatal day 35 rats was established, and single and repeat TBI (1-day interval) were examined histologically for axonal injury and behaviorally by the novel object recognition (NOR) task. The results from the current study demonstrate that an experimental closed head injury in the rodent with low mortality rates and absence of gross pathology can produce measurable cognitive deficits in a juvenile age group. The introduction of a second injury 24 h after the first impact resulted in increased axonal injury, astrocytic reactivity and increased memory impairment in the NOR task. The histological evidence demonstrates the potential usefulness of this RTBI model for studying the impact and time course of RTBI as it relates to the pediatric and young adult population. This study marks the first critical step in experimentally addressing the consequences of concussions and the cumulative effects of RTBI in the developing brain.

Acute neuroprotection to pilocarpine-induced seizures is not sustained after traumatic brain injury in the developing rat.

Following CNS injury there is a period of vulnerability when cells will not easily tolerate a secondary insult. However recent studies have shown that following traumatic brain injury (TBI), as well as hypoxic-ischemic injuries, the CNS may experience a period of protection termed "preconditioning." While there is literature characterizing the properties of vulnerability and preconditioning in the adult rodent, there is an absence of comparable literature in the developing rat. To determine if there is a window of vulnerability in the developing rat, post-natal day 19 animals were subjected to a severe lateral fluid percussion injury followed by pilocarpine (Pc)-induced status epilepticus at 1, 6 or 24 h post TBI. During the first 24 h after TBI, the dorsal hippocampus exhibited less status epilepticus-induced cell death than that normally seen following Pc administration alone. Instead of producing a state of hippocampal vulnerability to activation, TBI produced a state of neuroprotection. However, in a second group of animals evaluated 20 weeks post injury, double-injured animals were statistically indistinguishable in terms of seizure threshold, mossy fiber sprouting and cell survival when compared to those treated with Pc alone. TBI, therefore, produced a temporary state of neuroprotection from seizure-induced cell death in the developing rat; however, this ultimately conferred no long-term protection from altered hippocampal circuit rearrangements, enhanced excitability or later convulsive seizures.

Induction of monocarboxylate transporter 2 expression and ketone transport following traumatic brain injury in juvenile and adult rats.

Based on recent work demonstrating age-dependent ketogenic neuroprotection after traumatic brain injury (TBI), it was hypothesized that the neuroprotection among early post-weaned animals was related to induced cerebral transport of ketones after injury. Regional changes in monocarboxylate transporter 2 (MCT2) were acutely examined with immunohistochemistry after sham surgery or controlled cortical impact injury among postnatal day 35 and adult rats. Both ages showed elevated MCT2 expression in the ipsilateral cerebral vasculature after TBI. Using Western blotting, MCT2 expression was 80-88% greater in microvessels isolated from postnatal day 35 rats at all time points relative to adults. The increased MCT2 expression was temporally correlated with an age-related increase in cerebral uptake of ketones, when ketones were made available after injury.

Injury-induced alterations in N-methyl-D-aspartate receptor subunit composition contribute to prolonged 45calcium accumulation following lateral fluid percussion.

Cells that survive traumatic brain injury are exposed to changes in their neurochemical environment. One of these changes is a prolonged (48 h) uptake of calcium which, by itself, is not lethal. The N-methyl-D-aspartate receptor (NMDAR) is responsible for the acute membrane flux of calcium following trauma; however, it is unclear if it is involved in a flux lasting 2 days. We proposed that traumatic brain injury induced a molecular change in the NMDAR by modifying the concentrations of its corresponding subunits (NR1 and NR2). Changing these subunits could result in a receptor being more sensitive to glutamate and prolong its opening, thereby exposing cells to a sustained flux of calcium. To test this hypothesis, adult rats were subjected to a lateral fluid percussion brain injury and the NR1, NR2A and NR2B subunits measured within different regions. Although little change was seen in NR1, both NR2 subunits decreased nearly 50% compared with controls, particularly within the ipsilateral cerebral cortex. This decrease was sustained for 4 days with levels returning to control values by 2 weeks. However, this decrease was not the same for both subunits, resulting in a decrease (over 30%) in the NR2A:NR2B ratio indicating that the NMDAR had temporarily become more sensitive to glutamate and would remain open longer once activated. Combining these regional and temporal findings with 45calcium autoradiographic studies revealed that the degree of change in the subunit ratio corresponded to the extent of calcium accumulation. Finally, utilizing a combination of NMDAR and NR2B-specific antagonists it was determined that as much at 85% of the long term NMDAR-mediated calcium flux occurs through receptors whose subunits favor the NR2B subunit. These data indicate that TBI induces molecular changes within the NMDAR, contributing to the cells' post-injury vulnerability to glutamatergic stimulation.

Inhibition of neocortical plasticity during development by a moderate concussive brain injury.

To determine if a moderate traumatic brain injury (TBI) sustained early in life alters the capacity for developmental plasticity, 17-20-day-old rat pups received a lateral fluid percussion and then reared in an enriched environment for 17 days. Compared to sham-injured controls, this moderate TBI prevented the increase in cortical thickness (1.48 vs. 1.68 mm, p < 0.01) as well as the corresponding enhancement in cognitive performance in the Morris Water Maze (39 vs. 25 trials to criterion, p < 0.05). These injured animals exhibited no significant neuronal degeneration and no evidence of neurologic or motor deficits. These findings strongly support the conclusion that a diffuse brain injury is capable of inhibiting both anatomical and cognitive manifestations of experience-dependent developmental plasticity.

Neurotrophin dependence domain: a domain required for the mediation of apoptosis by the p75 neurotrophin receptor.

The mechanisms underlying neurotrophin dependence, and cellular dependent states in general, are unknown. We show that a 29 amino acid region in the intracellular domain of the common neurotrophin receptor, p75NTR, is required for the mediation of apoptosis by p75NTR. Furthermore, contrary to results obtained with Fas, monomeric p75NTR is required for apoptosis induction, whereas multimerization inhibits the pro-apoptotic effect. Within the 29-residue domain required for apoptosis induction by p75NTR, a 14-residue region is sufficient as a peptide inducer of apoptosis. This 14-residue peptide requires the positively charged carboxyterminal residues for its effect on cell death, and these same residues are required by the full-length p75NTR. These studies define a novel type of domain that mediates neurotrophin dependence, and suggest that other cellular dependent states may be mediated by proteins displaying similar domains.

Postoperative seizure control and antiepileptic drug use in pediatric epilepsy surgery patients: the UCLA experience, 1986-1997.

PURPOSE: Young children with refractory symptomatic epilepsy are at risk for developing neurologic and cognitive disabilities. Stopping the seizures may prevent these disabilities, but it is unclear whether resective surgery is associated with adequate long-term seizure control. METHODS: This study determined pre- and postsurgery seizure frequency and antiepileptic drug (AED) use (6 months to 10 years) in children with symptomatic seizures from unilateral cortical dysplasia (CD; n = 64) and non-CD etiologies (i.e., ischemia, infection; n = 71), and compared them with older temporal lobe epilepsy (TLE; n = 31) patients with complex partial seizures. RESULTS: Compared with presurgery, postsurgery seizure frequencies were decreased for CD, non-CD, and TLE patients (p < 0.002), and there were no differences between the three groups from 6 to 24 months after surgery (p > 0.12). At 5 years after surgery, seizure frequencies were greater in CD compared with TLE cases (p = 0.009). Compared with presurgery, the number of AEDs declined after surgery in all three groups (p < 0.002), and positively correlated with seizure frequencies (p = 0.0001). CONCLUSIONS: This study indicates that seizure relief and AED use after resective surgery for symptomatic CD and non-CD etiologies was comparable with complex partial TLE cases up to 2 years after surgery. Furthermore, at 5 years after surgery, CD patients had outcomes better than those before surgery, but worse than TLE cases. In young children, these findings support the concept that early removal of symptomatic pathologic substrates is associated with seizure control and reduced AED use, similar to that noted in older TLE cases up to 2 years after surgery. Seizure control may reduce the risk of developing the seizure-related encephalopathy associated with severe symptomatic early-onset childhood epilepsy.

Periventricular nodular heterotopia and childhood absence epilepsy.

A young female presented with an epileptic syndrome resembling childhood absence epilepsy, a normal neurologic examination, generalized 3-Hz spike-and-wave discharges, and clinical absences. Her seizures responded to treatment with valproic acid. Other abnormalities in her electroencephalogram prompted neuroimaging studies, which demonstrated periventricular nodular heterotopia. Review of published reports confirmed this presentation to be atypical of this developmental lesion. The authors describe their patient and discuss this unexpected association and the relevant reports briefly.

Pathogenesis of the developmental epilepsies.

The developmental epilepsies are distinctive in that they occur in a dynamic and plastic substrate. A variety of acquired insults may present with remarkably similar seizure syndromes that are age-specific and evolve with time. This evolution may be a consequence of the brain injury itself or an alteration of normal brain maturation due to an unfavorable electrical environment. Idiopathic epilepsies are now being correlated with specific gene defects, particularly those involving ion channels and/or neurotransmitter receptors. Despite this specificity, idiopathic seizure syndromes may be caused by mutations in different genes or different mutations in the same gene, and particular syndromes may manifest heterogeneous clinical seizure types. Not all localization-dependent epilepsies are symptomatic, as a growing number of genes have been identified with partial seizure syndromes. Febrile seizures probably do not represent a homogeneous entity, but multiple disorders that may be associated with developmental abnormalities and various coexisting seizure types.

High efficiency transient expression of eukaryotic genes: use of an HSV-1 immediate early promoter (ICP4).

We have compared the transcriptional efficiencies of a number of eukaryotic promoters following DNA-mediated transfection into cultured rat hepatoma cells. We find that the highest levels of expression for the bacterial chloramphenicol acetyltransferase (CAT) reporter gene are observed with a herpes simplex virus type 1 (HSV-1) immediate early promoter when co-transfected with an expression construct bearing the gene for the HSV-1 transcriptional activator protein VP16. This transactivation phenomenon is specific for the HSV-1 immediate early promoter and increases the expression of the reporter gene 7-fold. Expression from the ICP4 promoter is 2.5-fold greater than the other promoters tested. In addition, expression from the ICP4 promoter can be induced, at varying times following transfection, by infecting the cells with HSV-1 viral particles. Two plasmids have been constructed which contain the HSV-1 ICP4 promoter adjacent to a multiple cloning site. One of the plasmids also contains SV40 splicing and polyadenylation signals.