The Role of Astrogliosis in Formation of the Syrinx in Spinal Cord Injury.

A massive localized trauma to the spinal cord results in complex pathologic events driven by necrosis and vascular damage which in turn leads to hemorrhage and edema. Severe, destructive and very protracted inflammatory response is characterized by infiltration by phagocytic macrophages of a site of injury which is converted into a cavity of injury (COI) surrounded by astroglial reaction mounted by the spinal cord. The tissue response to the spinal cord injury (SCI) has been poorly understood but the final outcome appears to be a mature syrinx filled with the cerebrospinal fluid with related neural tissue loss and permanent neurologic deficits. This paper reviews known pathologic mechanisms involved in the formation of the COI after SCI and discusses the integrative role of reactive astrogliosis in mechanisms involved in the removal of edema after the injury. A large proportion of edema fluid originating from the trauma and then from vasogenic edema related to persistent severe inflammation, may be moved into the COI in an active process involving astrogliosis and specifically over-expressed aquaporins.

Neurologic and Histologic Tests Used to Measure Neuroprotective Effectiveness of Virus-Derived Immune-Modulating Proteins.

Severe inflammatory disease initiated by neurotrauma and stroke is of primary concern in these intractable pathologies as noted in recent studies and understanding of the pathogenesis of spinal cord injury (SCI) in the rat model. Successful anti-inflammatory treatments should result in neuroprotection and limit the loss of neurological function to injury caused by the initial damage. Continuous subdural infusion offers direct access to the cavity of injury (COI) that forms after balloon crush SCI deep in the spinal cord. Some anti-inflammatory compounds are not likely capable of crossing the blood-spinal cord barrier. Subdural infusion of myxoma virus-derived Serp-1, an anti-thrombotic/anti-thrombolytic, and also of M-T7, a chemokine inhibitor, improved the locomotor scores and pain sensation scores as well as reduced the numbers of macrophages in the COI by 50 and 80%, respectively, while intraperitoneal infusion of either protein had little effect. Injection of a chitosan hydrogel loaded with Serp-1 into the dorsal spinal column crush also resulted in improved neurological deficits and in reduction of the size of the crush lesion 4 weeks after injury. While neurological scores in a simplified hind-end (HE) locomotor test together with a toe-pinch withdrawal test demonstrated improvement in all balloon crush injury and dorsal spinal crush injury rats, a severe inflammation is induced by the injury indicating additional damage to the spinal cord. Thus neurological function testing can be contradictory, rather than corresponding, to the pathogenesis of SCI. The count of macrophages in the COI offers a precise, reliable method of measuring the effectiveness of a neuroprotective treatment of SCI in preclinical studies.

Neuroprotective Effect of Subdural Infusion of Serp-1 in Spinal Cord Trauma.

Spinal cord injury (SCI) initiates a severe, destructive inflammation with pro-inflammatory, CD68+/CD163-, phagocytic macrophages infiltrating the area of necrosis and hemorrhage by day 3 and persisting for the next 16 weeks. Inhibition of macrophage infiltration of the site of necrosis that is converted into a cavity of injury (COI) during the first week post-SCI, should limit inflammatory damage, shorten its duration and result in neuroprotection. By sustained subdural infusion we administered Serp-1, a Myxoma virus-derived immunomodulatory protein previously shown to improve neurologic deficits and inhibit macrophage infiltration in the COI in rats with the balloon crush SCI. Firstly, in a 7 day long study, we determined that the optimal dose for macrophage inhibition was 0.2 mg/week. Then, we demonstrated that a continuous subdural infusion of Serp-1 for 8 weeks resulted in consistently accelerated lowering of pro-inflammatory macrophages in the COI and in their almost complete elimination similar to that previously observed at 16 weeks in untreated SCI rats. The macrophage count in the COI is a quantitative test directly related to the severity of destructive inflammation initiated by the SCI. This test has consistently demonstrated anti-inflammatory effect of Serp-1 interpreted as neuroprotection, the first and necessary step in a therapeutic strategy in neurotrauma.

BMI1 is a therapeutic target in recurrent medulloblastoma.

Medulloblastoma (MB) is the most frequent malignant pediatric brain tumor, representing 20% of newly diagnosed childhood central nervous system malignancies. Although advances in multimodal therapy yielded a 5-year survivorship of 80%, MB still accounts for the leading cause of childhood cancer mortality. In this work, we describe the epigenetic regulator BMI1 as a novel therapeutic target for the treatment of recurrent human Group 3 MB, a childhood brain tumor for which there is virtually no treatment option beyond palliation. Current clinical trials for recurrent MB patients based on genomic profiles of primary, treatment-naive tumors will provide limited clinical benefit since recurrent metastatic MBs are highly genetically divergent from their primary tumor. Using a small molecule inhibitor against BMI1, PTC-028, we were able to demonstrate complete ablation of self-renewal of MB stem cells in vitro. When administered to mice xenografted with patient tumors, we observed significant reduction in tumor burden in both local and metastatic compartments and subsequent increased survival, without neurotoxicity. Strikingly, serial in vivo re-transplantation assays demonstrated a marked reduction in tumor initiation ability of recurrent MB cells upon re-transplantation of PTC-028-treated cells into secondary recipient mouse brains. As Group 3 MB is often metastatic and uniformly fatal at recurrence, with no current or planned trials of targeted therapy, an efficacious targeted agent would be rapidly transitioned to clinical trials.

Osseointegration of a 3D Printed Stemmed Titanium Dental Implant: A Pilot Study.

In this pilot study, a 3D printed Grade V titanium dental implant with a novel dual-stemmed design was investigated for its biocompatibility in vivo. Both dual-stemmed (n = 12) and conventional stainless steel conical (n = 4) implants were inserted into the tibial metaphysis of New Zealand white rabbits for 3 and 12 weeks and then retrieved with the surrounding bone, fixed, dehydrated, and embedded into epoxy resin. The implants were analyzed using correlative histology, microcomputed tomography, scanning electron microscopy (SEM), and transmission electron microscopy (TEM). The histological presence of multinucleated osteoclasts and cuboidal osteoblasts revealed active bone remodeling in the stemmed implant starting at 3 weeks and by 12 weeks in the conventional implant. Bone-implant contact values indicated that the stemmed implants supported bone growth along the implant from the coronal crest at both 3- and 12-week time periods and showed bone growth into microporosities of the 3D printed surface after 12 weeks. In some cases, new bone formation was noted in between the stems of the device. Conventional implants showed mechanical interlocking but did have indications of stress cracking and bone debris. This study demonstrates the comparable biocompatibility of these 3D printed stemmed implants in rabbits up to 12 weeks.

An in vivo model of anti-inflammatory activity of subdural dexamethasone following the spinal cord injury.

Current therapies to limit the neural tissue destruction following the spinal cord injury are not effective. Our recent studies indicate that the injury to the white matter of the spinal cord results in a severe inflammatory response where macrophages phagocytize damaged myelin and the fluid-filled cavity of injury extends in size with concurrent and irreversible destruction of the surrounding neural tissue over several months. We previously established that a high dose of 4mg/rat of dexamethasone administered for 1 week via subdural infusion remarkably lowers the numbers of infiltrating macrophages leaving large amounts of un-phagocytized myelin debris and therefore inhibits the severity of inflammation and related tissue destruction. But this dose was potently toxic to the rats. In the present study the lower doses of dexamethasone, 0.125-2.0mg, were administered via the subdural infusion for 2 weeks after an epidural balloon crush of the mid-thoracic spinal cord. The spinal cord cross-sections were analyzed histologically. Levels of dexamethasone used in the current study had no systemic toxic effect and limited phagocytosis of myelin debris by macrophages in the lesion cavity. The subdural infusion with 0.125-2.0mg dexamethasone over 2 week period did not eliminate the inflammatory process indicating the need for a longer period of infusion to do so. However, this treatment has probably lead to inhibition of the tissue destruction by the severe, prolonged inflammatory process.

Endpoints in myelin-deficient (MD) rats.

Health problems in some animal models remain unexplained, rendering in vivo studies ethically challenging, especially when experimental animals are prone to sudden death. Over the last 3 decades, the myelin-deficient (md) rat, a strain with severe dysmyelination due to mutant proteolipid protein, has been key to important discoveries in mechanisms of myelination and glial cell biology. The usefulness of this mutant rat, however, has been limited by sudden death during the fourth week of life. Timely euthanasia has been difficult because the cause of these mortalities remains unexplained and the endpoint not determined. In this clinicopathologic study, we determined that sudden onset of hindlimb paralysis inevitably leads to paralysis of the urinary bladder and then breathing difficulties because of severe injury to the spinal cord in the midthoracic region with concurrent narrowing of the vertebral canal due to fracture of a vertebral body. Sudden onset of hindlimb paralysis likely is related to seizures and severe muscle spasms that begin to occur at the end of the third week of life. Once seizure activity begins, we recommend frequent monitoring of md rats for hindlimb paralysis and distention of the urinary bladder as indication of endpoints mandating prompt euthanasia.

Eradication of Helicobacter spp. from a rat breeding colony.

Although Helicobacter spp. have been viewed as bacteria with low pathogenicity, many investigators have shown that these low-grade pathogens have the potential to become a severe threat in immunocompromised, inbred, and transgenic animals. Therefore the presence of Helicobacter spp. in experimental animals is considered to be an unacceptable variable. In this study a formulation of medicated feed was designed and tested in an attempt to eradicate Helicobacter spp. from an infected rat breeding colony. Two feeding protocols were used: 1) treating Helicobacter-infected pregnant dams to produce clean offspring and 2) treating infected adult animals long enough to eliminate the organisms. Bacterial DNA was extracted from feces and amplified using primers that recognized the Helicobacter spp.-specific region of the 16S rRNA gene. Fecal samples from the weanlings from protocol 1 tested negative for Helicobacter spp. at 1 week before and 2 and 12 weeks after weaning. Infected adult rats from protocol 2 tested negative after three cycles of 2 weeks on and 2 weeks off the medicated feed. Animals from both protocols have remained Helicobacter-free for 8 months.

Delivery of recombinant gene product to canine brain with the use of microencapsulation.

An alternative approach to somatic gene therapy is to deliver a therapeutic protein by implanting "universal" recombinant cells that are immunologically protected from graft rejection with alginate microcapsules. This strategy has proved successful in reversing pathologic conditions in several rodent models of human disease (dwarfism, lysosomal storage disease, hemophilia, cancer). In particular, neurologic disease and behavioral deficit in the mouse model of a neurodegenerative disease (mucopolysaccharidosis [MPS] VII) were significantly improved through the intraventricular implantation of the recombinant encapsulated cells. Here we report the feasibility of delivering recombinant gene products to the central nervous systems (CNSs) of dogs, first using human growth hormone as a marker for delivery in normal dogs and then using alpha-iduronidase as a therapeutic product for delivery in the MPS I dog that is genetically deficient in this lysosomal enzyme. Madin-Darby canine kidney cells were genetically modified to express either human growth hormone or canine alpha-iduronidase, then enclosed in alginate-poly-l-lysine-alginate microcapsules of about 500 microm in diameter. The encapsulated cells were implanted into the brain under steoreotaxic guidance. The brains were monitored with computed tomographic scans before and after surgery and examined biochemically and histologically. Delivery of gene products, as measured in the plasma and cerebrospinal fluid sampled periodically through 21 days or in various regions of the brain after death showed that the delivery of both gene products was extremely low but detectable. However, we noted extensive inflammatory reactions, both at the sites of implantation and in the immediate vicinity of the implanted microcapsules. Hence for this technology to be applicable to the CNSs of larger animals and human beings, a more accurate and less invasive neurosurgical procedure, more biocompatible microcapsule-recombinant cell combinations, and higher output of recombinant products must be developed.