Glial Associated Impairment of the Glymphatic System in Experimental Neonatal Hydrocephalus

Background Changes in aquaporin-4 (AQP4) and glial fibrillary acid protein (GFAP) expression by astrocytes have been observed in several pathologies. It is hypothesized that prolonged exposure to pathologically elevated intracranial pressure (ICP) may be linked to impaired glymphatic pathways. In this study we explore histological consequences of prolonged pressure-induced injury in a feline model of neonatal hydrocephalus through changes in AQP4 and GFAP expression. We discuss the implications this may have in gaining a better understanding of the underlying mechanisms of hydrocephalus (HCP). Methods Using a neonatal feline model, obstructive HCP was induced through kaolin injection into the cisterna magna. Time between injection and intervention via ventricular reservoir placement was used to divide groups into early and late treatment groups. Early and late animals received reservoirs at 1- and 2-weeks post kaolin injection, respectively. Controls underwent sham operations (saline injection instead of kaolin). Animals were sacrificed at 4 months allowing for a chronic treated hydrocephalic model at time of brain harvest. Immunofluorescent staining for GFAP, AQP4 and DAPI was performed on histological brain sections from each group, and densitometry was used to quantify the relative signal of protein expression. Results Hydrocephalus was seen in all animals receiving kaolin injection as demonstrated by magnetic resonance imaging, clinical examination and neurological sequelae. Hydrocephalic animals demonstrated lower levels of perivascular AQP4 expression, increased diffuse AQP4 expression and increased glial scarring of perivascular, ependymal and subependymal spaces. Cerebral microvasculature of early treatment groups demonstrated increased astrocytic processes in the perivascular spaces, while late treatment groups demonstrated increased glial scar formation. Overall, the glymphatic system was severely disrupted in chronic treated hydrocephalus compared to controls. Conclusions Reactive astrogliosis and AQP4 mislocation are evident in early and late reservoir-treated HCP. Glial scarring in the perivascular, ependymal and subependymal spaces concurrent with AQP4 internalization from the perivascular region are prominent in HCP conditions present within the neonatal period. Delay in treatment by 1 week demonstrates quantifiable increases in perivascular and ependymal glial scarring at 4 months of age. Further investigation is needed to correlate glymphatic disruption with impaired CSF absorption and its role in promoting progressive hydrocephalus.

Radiological, biomechanical and histopathological characteristics of rat brain in Kaolin-induced hydrocephalus

Objective: A better understanding of the pathophysiology and underlying mechanisms of brain damage in hydrocephalus is vital in developing diagnostic, observational and treatment tools that will have an impact on hydrocephalus outcomes. In this study, we aimed to demonstrate the radiological, biomechanical and histopathological characteristics of rat brain tissue in an experimental hydrocephalus model. Materials and Methods: Thirty-six male Sprague-Dawley rats (21 days old, weighing between 150 and 200 grams) were used in this study. Animals were randomly assigned to control (n = 6), 1-week hydrocephalus (n = 10), 2-week hydrocephalus (n = 10) and 3-week hydrocephalus (n = 10) groups. Hydrocephalus was induced with cisternal kaolin injection and controls received sham injection. Magnetic resonance imaging was used to measure ventricle size and cortical thickness. Vital signs, cerebral blood flow (CBF), mechanical tests and brain histology were assessed. Results: Three rats in the hydrocephalus group died during the follow-up, yielding an overall mortality of 10% among animals from hydrocephalus groups. Ventricular width, cross-sectional area of the lateral ventricles, ventricular index and ventricle / brain area ratio progressively increased and cortical thickness progressively decreased following kaolin injection. CBF was significantly lower at baseline than at 1st, 2nd and 3rd week (p < 0.05, for all). ICP was significantly elevated in all hydrocephalic groups in comparison with controls. EIT that was calculated from the first load-unload indentation test showed a significant increase at 2nd week post-injection (p=0.0001), indicating increased intracranial stiffness. However, this significant difference disappeared at 3rd week (p=0.956). Quantitative immunohistochemistry showed that hydrocephalic brains demonstrated significantly less NeuN-positive cells and significantly higher IBA-1-positive microglia and glial fibrillary acidic protein positive astrocytes cells in the cortex. Discussion and Conclusion: Cisternal kaolin injection causes varying degrees of ventricular enlargement in a rat model and hydrocephalus might contribute to neuronal and axonal damage and alter brain stiffness through axonal stretching or local hypoperfusion progressively over a period of days to months. As shown in this study, irreversible changes in viscoelastic behaviour and cellular structure develop in the late stages of hydrocephalus, suggesting the importance of early intervention in the treatment of hydrocephalus.

Cervical Central Canal Occlusion Induces Noncommunicating Syringomyelia

BACKGROUND: Mechanisms underlying the development of noncommunicating syringomyelia are poorly understood. OBJECTIVE: To assess the influence of focal arachnoiditis and central canal (CC) occlusion (CCO) on the formation of noncommunicating syringomyelia in the adult rat cervical spinal cord. Expression of pericanalicular aquaporin-4 is also examined. METHODS: Sprague-Dawley rats were subjected to circumferential or dorsal arachnoiditis (n = 34). Rats undergoing CCO (n = 69) were divided into 4 groups: group A, kaolin injection at a single site in the dorsal columns near the CC; group B, kaolin injection at multiple sites in the dorsal columns near the CC; group C, saline injection at multiple sites in the dorsal columns near the CC; or group D, controls. Rats were killed at 1, 4, 8, and 12 weeks. The CC area and aquaporin-4 (AQP4) expression were measured at the level of maximal CC enlargement. RESULTS: Circumferential and dorsal arachnoiditis induced a mild increase in the CC area at 12 weeks. Single-site CCO induced slight CC enlargement. In contrast, multiple sites of CCO in proximity frequently induced a major expansion of the CC area (up to 50 times). Increased AQP4 expression was observed in pericanalicular astrocytes proportional to the degree of CC expansion. CONCLUSION: Multiple sites of CCO created a model of noncommunicating syringomyelia in adult rats. Increased astrocytic AQP4 expression was proportional to the degree of CC expansion. Modulation of aquaporin expression may be a novel target for therapeutic interventions to prevent syringomyelia.

Mechanistic insights into posttraumatic syringomyelia based on a novel in vivo animal model

Object Although posttraumatic syringomyelia (PTS) develops in up to 30% of patients after spinal cord injury (SCI), the pathophysiology of this debilitating complication is incompletely understood. To provide greater insight into the mechanisms of this degenerative sequela of SCI, the authors developed and characterized a novel model of PTS. Methods The spinal cords of 64 female Wistar rats were injured by 35-g modified aneurysm clip compression at the level of T6–7. Kaolin (5 μl of 500 mg/ml solution) was then injected into the subarachnoid space rostral to the site of the injury to induce inflammatory arachnoiditis in 22 rats. Control groups received SCI alone (in 21 rats), kaolin injection alone (in 15 rats), or laminectomy and durotomy alone without injury (sham surgery in 6 rats). Results The combination of SCI and subarachnoid kaolin injection resulted in a significantly greater syrinx formation and perilesional myelomalacia than SCI alone; SCI and kaolin injection significantly attenuated locomotor recovery and exacerbated neuropathic pain (mechanical allodynia) compared with SCI alone. We observed that combined SCI and kaolin injection significantly increased the number of terminal deoxytransferase-mediated deoxyuridine triphosphate nick-end labeled–positive cells at 7 days after injury (p < 0.05 compared with SCI alone) and resulted in a significantly greater extent of astrogliosis and macrophage/microglial-associated inflammation at the lesion (p < 0.05). Conclusions The combination of compressive/contusive SCI with induced arachnoiditis results in severe PTS and perilesional myelomalacia, which is associated with enhanced inflammation, astrogliosis, and apoptotic cell death. The development of delayed neurobehavioral deficits and neuropathic pain in this model accurately reflects the key pathological and clinical conditions of PTS in humans.

Reduced Local Cerebral Blood Flow in Periventricular White Matter in Experimental Neonatal Hydrocephalus—Restoration with CSF Shunting

The extent to which the reduction in CBF occurring in hydrocephalus is a primary or secondary event in the pathogenesis of the brain injury that ensues has not been clearly established. This is particularly true in neonatal hydrocephalus, where the disorder is most common, and where timing of the treatment of the developing nervous system is so important. We investigated the changes in local CBF (lCBF) in an animal model of severe progressive neonatal hydrocephalus before and after CSF shunting. Hydrocephalus was induced in 27 1-week-old kittens by percutaneous injection of 0.05 ml of 25% kaolin into the cisterna magna. Fourteen littermates acted as controls. The lCBF was measured by 14C-iodoantipyrine quantitative autoradiography after 1 week in 15 animals (8 hydrocephalic, 7 controls) and after 3 weeks in 26 animals (19 hydrocephalic, 7 controls) following induction of hydrocephalus. Twelve of the 3-week hydrocephalic group received a ventriculoperitoneal shunt 10 days following kaolin injection. At 1 week following induction of hydrocephalus, lCBF was globally reduced in cortical gray matter and white matter as well as deep subcortical structures. The maximum reduction was in the parietal white matter, to 37% of control levels. At 3 weeks a significant reduction in lCBF persisted only in the white matter (parietal, occipital, and corpus callosum; average, 42% of control levels), whereas cortical gray and deep subcortical structures had returned to normal levels spontaneously. lCBF was normal in 3-week hydrocephalic shunted animals in all areas. CSF shunting restores the fall in lCBF in the periventricular white matter in this model. These findings are consistent with previous studies in the same model demonstrating derangement of high-energy phosphate metabolism and white matter anaerobic glycolysis with hydrocephalus and resolution with shunting.

Anaerobic glycolysis preceding white-matter destruction in experimental neonatal hydrocephalus

✓ The metabolic changes in neonatal hydrocephalus that lead to permanent brain injury are not clearly defined, nor is the extent to which these changes can be prevented by a cerebrospinal fluid shunt. To clarify these processes, cerebral glucose utilization was examined using [14C]2-deoxyglucose autoradiography in 1-month-old kittens, kaolin-induced hydrocephalic littermates, and hydrocephalic kittens in which a ventriculoperitoneal shunt had been inserted 10 days after kaolin injection. The hydrocephalic kittens showed thinning of the cerebral mantle and an anterior-to-posterior gradient of enlargement of the ventricular system, with a ventricle:brain ratio of 24% for the frontal and 35% for the occipital horns compared with control (< 0.5%) and shunted (< 5%) animals. White matter in hydrocephalic animals was edematous. Myelination was delayed in the periventricular region and in the cores of the cerebral gyri. Glucose utilization in hydrocephalic and shunted animals was unchanged from control animals in all gray-matter regions examined. However, in hydrocephalic animals, the frontal white matter exhibited a significant increase in glucose utilization (25 µmol • 100 gm−1 • min−1) in the cores of gyri compared with normal surrounding white-matter values (14.8 µmol • 100 gm−1 • min−1). Very low values (mean 4 µmol • 100 gm−1 • min−1) were found in areas corresponding to severe white-matter edema, and these areas were surrounded by a halo of increased activity (24 µmol • 100 gm−1 • min−1). In contrast, cytochrome oxidase activity in white matter was homogeneous. Shunting resulted in restoration of the cerebral mantle thickness, a return to normal levels of glucose utilization in the white matter, and an improvement in myelination. It is suggested that the areas of increased glucose utilization seen in the white matter represent anaerobic glycolysis which, if untreated, progresses to infarction. The pattern of this increased glucose utilization matches that of expected myelination and, during this period of high energy demand, white matter may be susceptible to the hypoperfusion associated with hydrocephalus.