Fundamental questions related to the locations of Cerebrospinal Spinal Fluid (CSF) absorption deficit and causes of the pressure gradients that expand the ventricles with hydrocephalus remain largely unanswered. Work in the Johnston lab over a 15 year period has demonstrated that CSF moves through the cribriform plate foramina in association with the olfactory nerves and is absorbed by a network of lymphatic vessels located within the olfactory turbinates. A kaolin-based rat model of communicating hydrocephalus was developed as a collaborative effort with Drs. McAllister, Wagshul and Li. After developing a method to quantify lymphatic CSF uptake in rats, we examined and observed that the movement of a radioactive tracer into the nasal turbinates was significantly reduced in the kaolin-injected animals compared to saline injected controls. However, it was possible that while lymphatic CSF uptake was compromised, other CSF absorption pathways may have compensated. To answer this, we measured the CSF outflow resistance (Rout) and observed it to be significantly greater in the kaolin group compared with animals receiving saline and there was a significant positive correlation between CSF Rout and ventricular volume. Nonetheless, it is not clear how impaired CSF clearance could lead to a dilation of the ventricles since the ventricular and subarachnoid compartments are in communication with one another and pressure would likely increase equally in both. At this point, we came across a theoretical paper that postulated that a drop in periventricular interstitial fluid pressure might provide an intraparenchymal pressure gradient favouring ventricular expansion. In addition, studies in non-CNS tissues indicated that a disruption of beta-1 (β1) integrin-matrix interactions could lower tissue pressure. Based on these suppositions and data, we examined if these concepts had relevance to the brain. For this, we measured pressure in the brain and observed a decline in periventricular pressures to values significantly below those monitored in the ventricular system following the injection of the anti integrin antibodies. Many of the animals developed hydrocephalus over 2 weeks post antibody injection. These data provide a novel mechanism for the generation of intraparenchymal pressure gradients that is likely contributing to ventricular expansion.

Diabetes mellitus is a complex disorder characterized by chronic hyperglycemia and vascular complications leading to significant morbidity and mortality. The common feature in all forms of diabetes is the insufficient functional β-cell mass to maintain euglycemia; therefore, the promotion of β-cell survival and growth is a fundamental goal for diabetes prevention and treatment. Evidence has suggested that erythropoietin (EPO) exerts cytoprotective effects on non-erythroid cells. However, the in vivo role of EPO on the pancreatic β cells has not been evaluated to date. We hypothesized that EPO would have direct cytoprotective effects on the pancreatic β cells and provide protection against experimental models of diabetes. In Chapter IV, we report that recombinant human erythropoietin (rHuEPO) administration provided protection against diabetes development in the streptozotocin (STZ)-induced and db/db mice, models of type 1 and type 2 diabetes, respectively, through anti-apoptotic, proliferative and angiogenic effects within the islets. Next, we show in Chapter V, using β cell-specific EPO-R and JAK2 knockout (KO) mice, that these cytoprotective effects of EPO resulted from direct biological effects on the β cells, and that JAK2 is its essential intracellular mediator. We also show that endogenous EPO or JAK2 in β cells had no essential role in determining β-cell development or homeostasis. Given that epo is a target gene of the hypoxia inducible factor (HIF) pathway, we hypothesized that deletion of von Hippel Lindau (VHL), a negative regulator of this pathway, in the β cells would lead to enhanced transcription of HIF-target genes, which are largely pro-survival, and lead to enhanced β-cell mass and function. Contrary to our hypothesis, in Chapter VI, our results show that the epo gene is not expressed in islets. Furthermore, β cell-specific VHL KO mice were glucose intolerant due to impaired β-cell function and mass, which we were able to rescue with rHuEPO treatment. Our results demonstrate that EPO exerts direct biological effects on the pancreatic β cells. Further understanding of the biology of EPO may hold promise for the development of a potential novel strategy for diabetes prevention and treatment.

Graduation date: 1992

Endoplasmic reticulum (ER) stress has been implicated in pancreatic beta-cell loss contributing to diabetes mellitus, however the molecular mechanisms of ER stress-induced apoptosis are unclear. In the first project of this thesis, the contribution of ER stress in proinflammatory cytokine-mediated beta-cell dysfunction and apoptosis is examined. Although exogenous cytokine treatment did induce unfolded protein response (UPR) genes, increased chaperone capacity had no effect on apoptosis induction, insulin biosynthesis and insulin secretion. Thus, ER stress is most likely not an important pathway in cytokine toxicity under our experimental system. The second project develops a pathophysiological model of ER stress based on the mutant misfolded insulin of the Akita mouse. Microarray analysis was conducted and we observed early induction of ER chaperone and ER-associated degradation (ERAD) genes, followed by a large increase in pro-apoptotic genes with mutant insulin expression. A detailed analysis of the ER stress response in this system is presented.

In order to elucidate how uncoupling protein 2 (UCP2) influences pancreatic β cells and glucose homeostasis, I have generated and characterized an inducible β cell-specific UCP2 deletion model,MIPCreER×loxUCP2 mice. Male littermates were injected with tamoxifen to induce UCP2 deletion(UCP2 iBKO) or with corn oil (CO). The phenotypes of both short-term (3-4 weeks after the last injection) and long-term (8-9 weeks after the last injection) were determined: Short-term iBKO mice displayed no differences in glucose or insulin tolerance, but enhanced in vivo and in vitro insulin secretion and suppressed islet reactive oxygen species (ROS) levels; while long-term iBKO mice displayed no difference in glucose tolerance, but impaired in vivo and in vitro insulin secretion and enhanced islet ROS levels. In conclusion, short-term UCP2 deletion in β cells promotes insulin secretion, while long-term UCP2 deletion impairs insulin secretion, possibly due to the opposite background of islet ROS.

Endoplasmic reticulum (ER) stress has been implicated in pancreatic beta-cell loss contributing to diabetes mellitus, however the molecular mechanisms of ER stress-induced apoptosis are unclear. In the first project of this thesis, the contribution of ER stress in proinflammatory cytokine-mediated beta-cell dysfunction and apoptosis is examined. Although exogenous cytokine treatment did induce unfolded protein response (UPR) genes, increased chaperone capacity had no effect on apoptosis induction, insulin biosynthesis and insulin secretion. Thus, ER stress is most likely not an important pathway in cytokine toxicity under our experimental system. The second project develops a pathophysiological model of ER stress based on the mutant misfolded insulin of the Akita mouse. Microarray analysis was conducted and we observed early induction of ER chaperone and ER-associated degradation (ERAD) genes, followed by a large increase in pro-apoptotic genes with mutant insulin expression. A detailed analysis of the ER stress response in this system is presented.

In order to elucidate how uncoupling protein 2 (UCP2) influences pancreatic β cells and glucose homeostasis, I have generated and characterized an inducible β cell-specific UCP2 deletion model,MIPCreER×loxUCP2 mice. Male littermates were injected with tamoxifen to induce UCP2 deletion(UCP2 iBKO) or with corn oil (CO). The phenotypes of both short-term (3-4 weeks after the last injection) and long-term (8-9 weeks after the last injection) were determined: Short-term iBKO mice displayed no differences in glucose or insulin tolerance, but enhanced in vivo and in vitro insulin secretion and suppressed islet reactive oxygen species (ROS) levels; while long-term iBKO mice displayed no difference in glucose tolerance, but impaired in vivo and in vitro insulin secretion and enhanced islet ROS levels. In conclusion, short-term UCP2 deletion in β cells promotes insulin secretion, while long-term UCP2 deletion impairs insulin secretion, possibly due to the opposite background of islet ROS.

The spatiotemporal dynamics of ongoing beta band (15-30 Hz) cortical oscillations and the modulation of this neural activity by tactile input and movement provide insight into how the brain achieves proper sensorimotor processing. Earlier studies have shown that the synchrony of the cortical beta rhythms within and between central and peripheral neuronal populations is modulated during and following somatosensation or movement, and correlated with effective motor control. In addition, abnormal levels of beta oscillations in the basal ganglia are correlated with motor dysfunction in Parkinson’s disease. Numerous functional roles for the beta rhythm have been proposed – ranging from inhibition to the facilitation of long-range communication. However, the neural network that generates the sensorimotor beta rhythm and the functional significance of this activity have not been fully specified. Thus, I used magnetoencephalography to complete three studies of the beta rhythm in healthy right-handed adults. In the first study, I hypothesized that finger vibration at beta frequencies would generate stimulus-coherent neuronal firing in the neural network that generates the beta rhythm – thus revealing the nodes of this network. Data were analyzed for nineteen subjects (10 females). The coherent activity was revealed using a novel analysis technique that generated whole-brain maps of inter-trial synchrony during passive repetitive finger vibration at 23 Hz. These maps identified contralateral primary somatosensory cortex (SI), posterior parietal cortex, supplementary motor area and primary motor cortex (MI), and ipsilateral brainstem as nodes in the network. In the second study, I correlated changes in focused attention with modulations in beta band cortical responses to specify the functional significance of this activity. Data were analyzed for twelve subjects (7 females). With increased focused attention to the stimulus, I hypothesized that the beta band responses to finger vibration would be enhanced in areas involved in somatosensory processing. A transient increase in the magnitude of beta oscillations in MI (event-related synchronization) following vibration offset was significantly enhanced by attention, as compared to passive stimulation. In addition, attention caused the suppression of beta oscillations (event-related desynchronization, ERD) in ipsilateral SI beginning 1 second prior to vibration offset. Strong attention-modulation of the beta rhythm outside of contralateral SI implies that these changes are indicative of higher-order processing of afferent information. In the third study, I tested the hypothesis that synchrony between beta rhythms in contralateral MI and the relevant muscle supports effective neuronal communication. I correlated changes in task performance with corticomuscular coherence (CMC) during the sustained application of force to match a visually-presented target. Data were analyzed for eighteen subjects (9 females). As predicted, CMC in MI was significantly increased during improved performance in this task. This suggests that central-peripheral synchrony plays an important functional role in sustaining isometric muscle control. Concurrent beta ERD in bilateral SI and primary visual cortices during the contraction indicates the importance of afferent feedback in this task. Gender-related effects were not investigated in these studies. Beta band neuromagnetic responses to movement and somatosensation identify a pervasive neural network that is involved in processing the relevant properties of somatic input and regulating sustained motor output.

Fundamental questions related to the locations of Cerebrospinal Spinal Fluid (CSF) absorption deficit and causes of the pressure gradients that expand the ventricles with hydrocephalus remain largely unanswered. Work in the Johnston lab over a 15 year period has demonstrated that CSF moves through the cribriform plate foramina in association with the olfactory nerves and is absorbed by a network of lymphatic vessels located within the olfactory turbinates. A kaolin-based rat model of communicating hydrocephalus was developed as a collaborative effort with Drs. McAllister, Wagshul and Li. After developing a method to quantify lymphatic CSF uptake in rats, we examined and observed that the movement of a radioactive tracer into the nasal turbinates was significantly reduced in the kaolin-injected animals compared to saline injected controls. However, it was possible that while lymphatic CSF uptake was compromised, other CSF absorption pathways may have compensated. To answer this, we measured the CSF outflow resistance (Rout) and observed it to be significantly greater in the kaolin group compared with animals receiving saline and there was a significant positive correlation between CSF Rout and ventricular volume. Nonetheless, it is not clear how impaired CSF clearance could lead to a dilation of the ventricles since the ventricular and subarachnoid compartments are in communication with one another and pressure would likely increase equally in both. At this point, we came across a theoretical paper that postulated that a drop in periventricular interstitial fluid pressure might provide an intraparenchymal pressure gradient favouring ventricular expansion. In addition, studies in non-CNS tissues indicated that a disruption of beta-1 (β1) integrin-matrix interactions could lower tissue pressure. Based on these suppositions and data, we examined if these concepts had relevance to the brain. For this, we measured pressure in the brain and observed a decline in periventricular pressures to values significantly below those monitored in the ventricular system following the injection of the anti integrin antibodies. Many of the animals developed hydrocephalus over 2 weeks post antibody injection. These data provide a novel mechanism for the generation of intraparenchymal pressure gradients that is likely contributing to ventricular expansion.

Diabetes mellitus is a complex disorder characterized by chronic hyperglycemia and vascular complications leading to significant morbidity and mortality. The common feature in all forms of diabetes is the insufficient functional β-cell mass to maintain euglycemia; therefore, the promotion of β-cell survival and growth is a fundamental goal for diabetes prevention and treatment. Evidence has suggested that erythropoietin (EPO) exerts cytoprotective effects on non-erythroid cells. However, the in vivo role of EPO on the pancreatic β cells has not been evaluated to date. We hypothesized that EPO would have direct cytoprotective effects on the pancreatic β cells and provide protection against experimental models of diabetes. In Chapter IV, we report that recombinant human erythropoietin (rHuEPO) administration provided protection against diabetes development in the streptozotocin (STZ)-induced and db/db mice, models of type 1 and type 2 diabetes, respectively, through anti-apoptotic, proliferative and angiogenic effects within the islets. Next, we show in Chapter V, using β cell-specific EPO-R and JAK2 knockout (KO) mice, that these cytoprotective effects of EPO resulted from direct biological effects on the β cells, and that JAK2 is its essential intracellular mediator. We also show that endogenous EPO or JAK2 in β cells had no essential role in determining β-cell development or homeostasis. Given that epo is a target gene of the hypoxia inducible factor (HIF) pathway, we hypothesized that deletion of von Hippel Lindau (VHL), a negative regulator of this pathway, in the β cells would lead to enhanced transcription of HIF-target genes, which are largely pro-survival, and lead to enhanced β-cell mass and function. Contrary to our hypothesis, in Chapter VI, our results show that the epo gene is not expressed in islets. Furthermore, β cell-specific VHL KO mice were glucose intolerant due to impaired β-cell function and mass, which we were able to rescue with rHuEPO treatment. Our results demonstrate that EPO exerts direct biological effects on the pancreatic β cells. Further understanding of the biology of EPO may hold promise for the development of a potential novel strategy for diabetes prevention and treatment.