Michael J. Whalen

Cell–cell Signaling in the Neurovascular Unit

Historically, the neuron has been the conceptual focus for almost all of neuroscience research. In recent years, however, the concept of the neurovascular unit has emerged as a new paradigm for investigating both physiology and pathology in the CNS. This concept proposes that a purely neurocentric focus is not sufficient, and emphasizes that all cell types in the brain including neuronal, glial and vascular components, must be examined in an integrated context. Cell–cell signaling and coupling between these different compartments form the basis for normal function. Disordered signaling and perturbed coupling form the basis for dysfunction and disease. In this mini-review, we will survey four examples of this phenomenon: hemodynamic neurovascular coupling linking blood flow to brain activity; cellular communications that evoke the blood–brain barrier phenotype; parallel systems that underlie both neurogenesis and angiogenesis in the CNS; and finally, the potential exchange of trophic factors that may link neuronal, glial and vascular homeostasis.

Necrostatin-1 Reduces Histopathology and Improves Functional Outcome after Controlled Cortical Impact in Mice:

Necroptosis is a newly identified type of programmed necrosis initiated by the activation of tumor necrosis factor alpha (TNFα)/Fas. Necrostatin-1 is a specific inhibitor of necroptosis that reduces ischemic tissue damage in experimental stroke models. We previously reported decreased tissue damage and improved functional outcome after controlled cortical impact (CCI) in mice deficient in TNFα and Fas. Hence, we hypothesized that necrostatin-1 would reduce histopathology and improve functional outcome after CCI in mice. Compared with vehicle-/inactive analog-treated controls, mice administered necrostatin-1 before CCI had decreased propidium iodide-positive cells in the injured cortex and dentate gyrus (6 h), decreased brain tissue damage (days 14, 35), improved motor (days 1 to 7), and Morris water maze performance (days 8 to 14) after CCI. Improved spatial memory was observed even when drug was administered 15 mins after CCI. Necrostatin-1 treatment did not reduce caspase-3-positive cells in the dentate gyrus or cortex, consistent with a known caspase-independent mechanism of necrostatin-1. However, necrostatin-1 reduced brain neutrophil influx and microglial activation at 48 h, suggesting a novel anti-inflammatory effect in traumatic brain injury (TBI). The data suggest that necroptosis plays a significant role in the pathogenesis of cell death and functional outcome after TBI and that necrostatin-1 may have therapeutic potential for patients with TBI.

Biochemical, cellular, and molecular mechanisms in the evolution of secondary damage after severe traumatic brain injury in infants and children: Lessons learned from the bedside.

Objective To present a state-of-the-art review of mechanisms of secondary injury in the evolution of damage after severe traumatic brain injury in infants and children. Data Sources We reviewed 152 peer-reviewed publications, 15 abstracts and proceedings, and other material relevant to the study of biochemical, cellular, and molecular mechanisms of damage in traumatic brain injury. Clinical studies of severe traumatic brain injury in infants and children were the focus, but reports in experimental models in immature animals were also considered. Results from both clinical studies in adults and models of traumatic brain injury in adult animals were presented for comparison. Data Synthesis Categories of mechanisms defined were those associated with ischemia, excitotoxicity, energy failure, and resultant cell death cascades; secondary cerebral swelling; axonal injury; and inflammation and regeneration. Conclusions A constellation of mediators of secondary damage, endogenous neuroprotection, repair, and regeneration are set into motion in the brain after severe traumatic injury. The quantitative contribution of each mediator to outcome, the interplay between these mediators, and the integration of these mechanistic findings with novel imaging methods, bedside physiology, outcome assessment, and therapeutic intervention remain an important target for future research.

Interleukin-8 is increased in cerebrospinal fluid of children with severe head injury.

Objective: To determine interleukin (IL)‐8 concentrations in ventricular cerebrospinal fluid from children with severe traumatic brain injury (TBI). Design: Prospective study. Setting: University childrens hospital. Patients: Twenty‐seven children hospitalized with severe TBI (Glasgow Coma Scale score ≤8), seven children with cerebrospinal fluid culture‐positive bacterial meningitis, and twenty‐four age‐equivalent controls. Interventions: Placement of an intraventricular catheter and continuous drainage of cerebrospinal fluid. Measurements and Main Results: Median [range] cerebrospinal fluid IL‐8 concentration in children with TBI (0‐12 hrs) (4,452.5 [0‐20,000] pg/mL) was markedly greater than that in controls (14.5 [0‐250]) (p < .0001) and equivalent to concentrations in children with meningitis (5,300 [1,510‐22,000] pg/mL) (p = .33). Cerebrospinal fluid IL‐8 remained increased in children with severe TBI for up to 108 hrs after injury. Univariate logistic regression analysis demonstrated an association between cerebrospinal fluid IL‐8 and child abuse (p = .07) and mortality (p = .01). Multivariate analysis demonstrated a strong, independent association between cerebrospinal fluid IL‐8 and mortality (p = .01). Conclusions: The data are consistent with an acute inflammatory component of TBI in children and suggest an association between cerebrospinal fluid IL‐8 and outcome after TBI. IL‐8 may represent a potential target for anti‐inflammatory therapy.

FGF-2 regulates neurogenesis and degeneration in the dentate gyrus after traumatic brain injury in mice

We studied the role of FGF-2 on regulation of neurogenesis and cell loss in the granule cell layer (GCL) of the hippocampal dentate gyrus after experimental traumatic brain injury (TBI). In both FGF-2(-/-) and FGF-2(+/+) mice subjected to controlled cortical impact, the number of dividing cells labeled with BrdU, injected on posttrauma days 6 through 8, increased at 9 days after TBI, and the number of BrdU-positive cells colabeled with neuron-specific nuclear antigen significantly increased at 35 days. However, in injured FGF-2-/- mice, BrdU-positive cells and BrdU-positive neurons (days 9, 35) were fewer compared with FGF-2(+/+) mice. There was also a decrease in the volume of the GCL and the number of GCL neurons after TBI in both FGF-2(-/-) and FGF-2(+/+) mice, but the decrease in both was greater in FGF-2-/- mice at 35 days. Overexpression of FGF-2 by intracerebral injection of herpes simplex virus-1 amplicon vectors encoding this factor increased numbers of dividing cells (day 9) and BrdU-positive neurons (day 35) significantly in C57BL/6 mice. Furthermore, the decrease in GCL volume was also attenuated. These results suggest that FGF-2 upregulates neurogenesis and protects neurons against degeneration in the adult hippocampus after TBI, and that FGF-2 supplementation via gene transfer can reduce GCL degeneration after TBI.

Reduction of cognitive and motor deficits after traumatic brain injury in mice deficient in poly(ADP-ribose) polymerase.

Poly(ADP-ribose) polymerase (PARP), or poly-(ADP-ribose) synthetase, is a nuclear enzyme that consumes NAD when activated by DNA damage. The role of PARP in the pathogenesis of traumatic brain injury (TBI) is unknown. Using a controlled cortical impact (CCI) model of TBI and mice deficient in PARP, the authors studied the effect of PARP on functional and histologic outcome after CCI using two protocols. In protocol 1, naïve mice (n = 7 +/+, n = 6 –/–) were evaluated for motor and memory acquisition before CCI. Mice were then subjected to severe CCI and killed at 24 hours for immunohistochemical detection of nitrated tyrosine, an indicator of peroxynitrite formation. Motor and memory performance did not differ between naïve PARP +/+ and –/– mice. Both groups showed nitrotyrosine staining in the contusion, suggesting that peroxynitrite is produced in contused brain. In protocol 2, mice (PARP +/+, n = 8; PARP –/–, n = 10) subjected to CCI were tested for motor and memory function, and contusion volume was determined by image analysis. PARP –/– mice demonstrated improved motor and memory function after CCI versus PARP +/+ mice (P < 0.05). However, contusion volume was not different between groups. The results suggest a detrimental effect of PARP on functional outcome after TBI.

TNF Alpha and Fas Mediate Tissue Damage and Functional Outcome after Traumatic Brain Injury in Mice

Tumor necrosis factor-alpha (TNFα) and Fas are induced after traumatic brain injury (TBI); however, their functional roles are incompletely understood. Using controlled cortical impact (CCI) and mice deficient in TNFα, Fas, or both (TNFα/Fas—/—), we hypothesized that TNFα and Fas receptor mediate secondary TBI in a redundant manner. Compared with wild type (WT), TNFα/Fas—/— mice had improved motor performance from 1 to 4 days (P < 0.05), improved spatial memory acquisition at 8 to 14 days (P < 0.05), and decreased brain lesion size at 2 and 6 weeks after CCI (P < 0.05). Protection in TNFα/Fas—/— mice from histopathological and motor deficits was reversed by reconstitution with recombinant TNFα before CCI, and TNFα—/— mice administered anti-Fas ligand antibodies had improved spatial memory acquisition versus similarly treated WT mice (P < 0.05). Tumor necrosis factor-alpha/Fas—/— mice had decreased the numbers of cortical cells with plasmalemma damage at 6h (P < 0.05 versus WT), and reduced matrix metalloproteinase-9 activity in injured brain at 48 and 72 h after CCI. In immature mice subjected to CCI, genetic inhibition of TNFα and Fas conferred beneficial effects on histopathology and spatial memory acquisition in adulthood (both P < 0.05 versus WT), suggesting that the beneficial effects of TNFα/Fas inhibition may be permanent. The data suggest that redundant signaling pathways initiated by TNFα and Fas play pivotal roles in the pathogenesis of TBI, and that biochemical mechanisms downstream of TNFα/Fas may be novel therapeutic targets to limit neurological sequelae in children and adults with severe TBI.

Clinical correlates in an experimental model of repetitive mild brain injury.

Although there is growing awareness of the long‐term cognitive effects of repetitive mild traumatic brain injury (rmTBI; eg, sports concussions), whether repeated concussions cause long‐term cognitive deficits remains controversial. Moreover, whether cognitive deficits depend on increased amyloid β deposition and tau phosphorylation or are worsened by the apolipoprotein E4 allele remains unknown. Here, we use an experimental model of rmTBI to address these clinical controversies.

Comparison of therapeutic effects between pulsed and continuous wave 810-nm wavelength laser irradiation for traumatic brain injury in mice.

Background and Objective Transcranial low-level laser therapy (LLLT) using near-infrared light can efficiently penetrate through the scalp and skull and could allow non-invasive treatment for traumatic brain injury (TBI). In the present study, we compared the therapeutic effect using 810-nm wavelength laser light in continuous and pulsed wave modes in a mouse model of TBI. Study Design/Materials and Methods TBI was induced by a controlled cortical-impact device and 4-hours post-TBI 1-group received a sham treatment and 3-groups received a single exposure to transcranial LLLT, either continuous wave or pulsed at 10-Hz or 100-Hz with a 50% duty cycle. An 810-nm Ga-Al-As diode laser delivered a spot with diameter of 1-cm onto the injured head with a power density of 50-mW/cm2 for 12-minutes giving a fluence of 36-J/cm2. Neurological severity score (NSS) and body weight were measured up to 4 weeks. Mice were sacrificed at 2, 15 and 28 days post-TBI and the lesion size was histologically analyzed. The quantity of ATP production in the brain tissue was determined immediately after laser irradiation. We examined the role of LLLT on the psychological state of the mice at 1 day and 4 weeks after TBI using tail suspension test and forced swim test. Results The 810-nm laser pulsed at 10-Hz was the most effective judged by improvement in NSS and body weight although the other laser regimens were also effective. The brain lesion volume of mice treated with 10-Hz pulsed-laser irradiation was significantly lower than control group at 15-days and 4-weeks post-TBI. Moreover, we found an antidepressant effect of LLLT at 4-weeks as shown by forced swim and tail suspension tests. Conclusion The therapeutic effect of LLLT for TBI with an 810-nm laser was more effective at 10-Hz pulse frequency than at CW and 100-Hz. This finding may provide a new insight into biological mechanisms of LLLT.

Blood Brain Barrier Permeability and Acute Inflammation in Two Models of Traumatic Brain Injury in the Immature Rat: A Preliminary Report

We sought to investigate the course and magnitude of blood brain barrier (BBB) permeability following focal and diffuse traumatic brain injury (TBI) in immature rats and examine the time course of markers of acute inflammation (neutrophil accumulation and E-selectin [E-sel] expression) following these two types of injury. We measured BBB permeability using i.v. injection Evans Blue (EB) and the extent of inflammation using immunohistochemical techniques identifying neutrophils (monoclonal antibody RP-3) and the endothelial adhesion molecule, E-selectin. Male Sprague-Dawley immature (17 day-old) rats (30-45 g, n = 80) were subjected to a controlled cortical impact (CCI: 2 mm, 4 m/s), a closed head diffuse injury (DI: 150 g/2m) or a corresponding sham procedure (with or without craniotomy). EB was injected i.v. at 30 min before sacrifice, which occurred at 1 h, 4 h, or 24 h after injury. BBB permeability was observed in both the CCI and DI rats at 1 h after injury which largely resolved by 24 h. In the CCI, EB extravasation was seen within and around the contusion. In DI, diffuse BBB permeability was seen. DI was not associated with acute inflammation since there was neither neutrophil accumulation nor E-selectin expression. The CCI rats though had 5.1 +/- 2.2 neutrophils/hpf and 3.0 +/- 0.4 endothelial cells/hpf expressing E-selectin (mean +/- SEM) (both p < 0.05 vs sham and DI). These data suggest that BBB breakdown occurs in the immature rat after both focal and diffuse TBI. This early BBB permeability was not associated with acute inflammation in DI but was in CCI. These data also suggest that contusion is a key factor in the development of a traditional acute inflammatory response after TBI in the immature rat.