David O. Okonkwo

Impaired axonal transport and altered axolemmal permeability occur in distinct populations of damaged axons following traumatic brain injury.

Traumatic axonal injury (TAI) evolves within minutes to hours following traumatic brain injury (TBI). Previous studies have identified axolemmal disruption and impaired axonal transport (AxT) as key mechanisms in the evolution of TAI. While initially hypothesized that axolemmal disruption culminates in impaired AxT, previous studies employed single-label methodologies that did not allow for a full determination of the spatial-temporal relationships of these two events. To explore directly the relationship between impaired AxT and altered axolemmal permeability, the current investigation employed 40, 10, and 3 kDa fluorescently conjugated dextrans as markers of axolemmal integrity, with antibodies targeting the anterogradely transported amyloid precursor protein (APP) utilized as a marker of impaired AxT. Rats underwent impact acceleration TBI and were intrathecally administered 40 kDa, 40 + 10 kDa or 40 + 3 kDa fluorescently tagged dextrans, with brains subsequently prepared for APP immunofluorescence. Brainstem corticospinal tracts (CSpT), medial lemnisci (ML), and medial longitudinal fasciculi were examined for evidence of TAI. APP and all dextrans consistently localized to distinct classes of TAI. Dextrans were noted as early as 5 min following injury within axonal segments demonstrating an irregular/tortuous appearance, and were seen within thin and elongate/vacuolated axons by 30 min-6 h following injury. APP, first noted within swollen axons at 30 min following injury, was found within progressively swollen axons that showed no dextran colocalization within 3 h of injury. However, by 6 h, dextrans colocalized in disconnected axonal bulbs. At this time-point, dextrans also persisted within single-labeled, highly vacuolated/thin, and elongate axons. These studies confirm that axolemmal disruption and impaired AxT occur as distinct non-related events early in the pathogenesis of TAI. Further, these studies provide evidence that the process of impaired axonal transport and subsequent axonal disconnection leads to delayed axolemmal instability, rather than proceeding as a consequence of initial axolemmal failure. This finding underscores the need of multiple approaches to fully assess the axonal response to TBI.

Clinical characteristics of silent corticotrophic adenomas and creation of an internet-accessible database to facilitate their multi-institutional study.

OBJECTIVESilent corticotrophic adenomas (SCAs) of the pituitary gland present as clinically nonfunctioning sellar lesions, with normal serum and urine hormone testing results, but stain positively for adrenocorticotropic hormone in immunohistochemical analyses. These tumors are now more readily recognized, but determination of their natural history and responses to treatment is difficult because of their rarity. We report the diagnoses and outcomes for a series of patients with SCAs, and we describe the creation of an Internet-accessible database (www.hsc.virginia.edu/neuro/neurosurgery/pituitary.html) for collection of multi-institutional data on these lesions. METHODSThe medical records of patients with documented SCAs who were treated at the University of Virginia between 1991 and 2002 were reviewed. A comprehensive data collection form was then created and posted online. RESULTSTwenty-seven patients with SCAs were identified, with a female predominance (70%, P = 0.04). Headache was the most common presenting symptom (70%), followed by visual field deficits (52%), acute or subacute pituitary apoplexy (33%), cavernous sinus syndrome (18.5%), and hypopituitarism (11.1%). Extrasellar extension was noted for 92.6% of patients on preoperative magnetic resonance imaging scans. Transsphenoidal surgery was performed for all patients. Follow-up information was available for all patients (median, 60 mo; range, 3–254 mo). Postoperatively, 33% of patients received radiotherapy. Recurrence was noted for 37% of all patients and 41.7% of patients who did not receive postoperative radiotherapy. CONCLUSIONSCAs, although clinically nonfunctioning, may behave like aggressive adrenocorticotropic hormone-secreting adenomas and therefore should receive vigorous follow-up monitoring, with consideration being given to the recommendation of radiotherapy in cases with residual tumor.

Illuminating protein interactions in tissue using confocal and two-photon excitation fluorescent resonance energy transfer microscopy

Traumatic brain injury (TBI) remains the most common cause of death in persons under age 45 in the Western world. One of the principal determinants of morbidity and mortality following TBI is traumatic axonal injury (TAI). Current hypotheses on the pathogenesis of TAI involve activation of apoptotic cascades secondary to TBI. While a number of studies have demonstrated direct evidence for the activation of apoptotic cascades in TAI, the precise pathway by which these cascades are initiated remains a subject of intense investigation. As axolemmal disruption with the subsequent intra-axonal influx of large molecular weight species has been demonstrated to occur in relation to local axonal breakdown, attention has focused on cascades that may occur as a result of loss of ionic homeostasis. One proposed pathway by which this has been hypothesized to occur is the Ca(2+)-mediated activation of calmodulin and subsequent activation of the phosphatase calcineurin with dephosphorylation of a protein known as BAD, leading to a proapoptotic interaction between BAD and the mitochondrial protein Bcl-xL. While this pathway is an intriguing route for traumatic axonal pathogenesis, neither conventional immunocytochemical/histochemical nor ultrastructural approaches have had the capacity to shed insight on whether BAD and Bcl-xL interact in TAI in vivo. We describe the implementation of confocal and two-photon excitation fluorescence resonance energy transfer (FRET) microscopy techniques through which we demonstrate interaction between the proapoptotic protein BAD and the prosurvival protein Bcl-xL within TAI following TBI. Further, we report on a method to reliably detect protein interactions within aldehyde fixed tissue sections through conventional immunohistochemical approaches.

Trans-sodium crocetinate increases oxygen delivery to brain parenchyma in rats on oxygen supplementation

Trans-sodium crocetinate (TSC) is a vitamin A-analog that increases diffusivity of oxygen in aqueous solutions, including plasma. The current study is the initial investigation of the effects of TSC on oxygen delivery to brain. Adult male rats were intubated and ventilated with 21%, 60%, or 100% oxygen. A craniotomy was performed and a Licox rat brain tissue PO(2) probe inserted into parietal cortex. Rats were then administered intravenous infusions of either TSC or saline and brain tissue PO(2) values were recorded. TSC significantly increased brain tissue oxygen delivery. This effect was minimal in rats ventilated with normal air and substantial in rats on oxygen supplementation. Arterial blood gas parameters did not differ within groups. These results provide clear indication to study the utility of TSC in ameliorating hypoxic/ischemic insults in neurological disorders.

Supratentorial dural-based hemangioblastoma not associated with von Hippel Lindau complex.

SummaryHemangioblastomas are rarely found in a supratentorial location and are commonly associated with the von Hippel-Lindau complex. Therefore, patients with such tumors must be evaluated for both other hemangioblastomas within the central nervous system as well as for this complex via physical examination, radiographic examination, and genetic testing. We report the seventh case of a patient with an isolated supratentorial dural based hemangioblastoma not associated with the von Hippel-Lindau complex.

Basic science of closed head injuries and spinal cord injuries.

Traumatic CNS injury is one of the most important health issues in our society and is a risk to all athletes, both in competitive and recreational sports. Our understanding of the pathophysiology has improved tremendously in the last 20 years. This progress has led to the identification of several possible treatments for improving outcome following spinal cord injury and traumatic brain injury. As no panacea exists, improvements in experimental models have empowered researchers in their search for novel therapeutic strategies.

Arachnoid diverticula associated with anterior cranial base tumors: technical case report.

INTRODUCTION Cerebrospinal fluid (CSF) diverticula are uncommonly associated with anterior cranial base tumors. When they occur, they often complicate the surgical management of these tumors via a transsphenoidal approach. This report examines the effectiveness of transsphenoidal surgery in the treatment of these rare entities. METHODS We performed a review of four sellar and parasellar tumors (three pituitary adenomas and one chordoma) that contained an arachnoid diverticulum communicating with the intracranial subarachnoid space. RESULTS One of these tumors (a prolactinoma) was successfully treated medically and regressed dramatically in size; the remaining three were treated by transsphenoidal resection. In all of the operative cases, the tumor was successfully resected with no endocrinological or neurological deficit. An intraoperative CSF leak was encountered in each operative case and was treated by packing the sella with an abdominal fat graft. One patient returned to the operating room for a CSF leak repair. No instances of infection, meningitis, or other major complications occurred. Sellar and parasellar tumors associated with arachnoid diverticula can be successfully treated using transsphenoidal surgery. CONCLUSION Preoperative anatomic localization of the CSF diverticulum, as well as anticipatory planning for an intraoperative CSF leak are essential. Whenever possible, it is probably wise to preserve the arachnoidocele rather than to rupture it intentionally, obliterating the dead space within the sella in either situation.

Lumbar Deformity (Vascular) Surgery Complication

Major vascular injury in spinal deformity surgery is a rare but potentially catastrophic complication. There is paucity of data on iatrogenic major vascular injuries during posterior spinal instrumentation procedures. Most surgeons would agree that the incidence of vascular injury in spine surgery is underreported. The aorta remains in close proximity to the spinal column even in the most deformed spines. Injuries to the aorta can occur in the perioperative period due to iatrogenic vessel injury or may manifest as secondary injuries as prominent implants erode this major vessel. We present the case of an 82-year-old woman who sustained an iatrogenic vascular injury when a misplaced lumbar pedicle screw perforated the aorta. The aortic perforation was at the origin of the renal arteries, necessitating open repair instead of endovascular management. We discuss the incidence and management of the potentially devastating complication of vascular injury in spinal reconstructive surgery.

6 – Multiphoton FRET Microscopy for Protein Localization in Tissue

This chapter discusses the multiphoton Forster resonance energy transfer (mp-FRET) microscopy and its use in localizing the proteins in tissue following traumatic brain injury (TBI).To localize proteins, two fluorophore molecules need to be attached to each protein molecule under investigation. One should have a good expression level of proteins with the selected fluorophores and use the best optics, a high quantum efficiency charge-coupled device (CCD) camera (for real-time 2p-FRET) or photomultiplier tubes (PMT) to acquire the FRET images. The efficiency of the FRET could also be improved by optimizing the concentration of the fluorophore used for protein labeling. The z-plane focus motor of the microscope to focus on the distal surface of the specimen is used to determine whether images acquired with the mp-FRET microscopy in thicker tissue specimens are appropriate for the FRET image analysis.

Multiphoton tissue FRET demonstrates in vivo BAD/Bcl-xL heterodimerization in injured axons following traumatic brain injury in rats

Traumatic brain injury (TBI) remains the most common cause of death in persons under age 45 in the Western world. A devastating element of TBI is the diffuse and widespread injury of axons in a process known as traumatic axonal injury (TAI). TAI is a difficult entity to study as gross protein or molecular analyses have been largely precluded due to the requisite for specifically localizing protein changes to axons rather than the supporting glia or neuronal soma. As such, much of the mechanistic insight into TAI pathogenesis thusfar has come through histochemical and immunohistochemical analyses. In order to address the next generation of questions in TAI pathogenesis it has become critical to develop methodologies that allow for direct examination of protein-protein interactions in relation to sites of TAI. In the current communication, we report on a modified method of multiphoton Fluorescent Resonance Energy Transfer (FRET) microscopy that allows for the direct assessment of protein-protein interactions in aldehyde fixed tissue sections through the use of a conventional dual-label immunofluorescent approach. In the utilization of this technique, we explored whether the bcl-2-related proteins BAD and Bcl-xL heterodimerize in a pro-apoptotic fashion within traumatically injured axons following TBI. Adult SD rats were subjected to impact acceleration TBI and euthanized at multiple time points. Vibratome sections derived from adult SD rats that had previously undergone impact acceleration TBI were processed for immunohistochemical double labeling with anti-BAD 1° antibody/Alexa 488 2° antibody, followed by anti-Bcl-xL 1° antibody/Alexa 555 2° antibody. Images were processed for spectral bleedthrough, and efficiency/distance calculations were performed. BAD/Bcl-xL heterodimerization was examined in relation to cytochrome c release and caspase-3 activation, by employing a third immunofluorescent label visualized with an Alexa 647 dye. Multiphoton FRET microscopy was carried out upon 40 micron thick sections. Further, to determine if FRET analysis could be performed in thick tissue specimens, 100 and 200 micron thick sections were examined as well. At 6h postinjury, swollen axons in medial lemniscus demonstrated a mean energy transfer efficiency greater than 20% indicating formation of Bad-Bcl-xL complexes. Thick tissue specimens up to 200 microns thick likewise demonstrated FRET efficiencies greater than 20%. Specimens positively labeled for either cytochrome c or caspase-3 demonstrated FRET efficiencies greater than 10%. The current investigation demonstrates FRET microscopy can be employed to assess protein-protein interactions in aldehyde-fixed tissue sections through multi-label immunofluorescent methodologies. It is believed that the current approach will have widespread applicability as examination of protein-protein interactions within in vivo systems may now be assessed on the cellular and subcellular level within aldehyde fixed tissue sections.