Paolo Bazzigaluppi

Early neurovascular dysfunction in a transgenic rat model of Alzheimer’s disease

Alzheimer’s disease (AD), pathologically characterized by amyloid-β peptide (Aβ) accumulation, neurofibrillary tangle formation, and neurodegeneration, is thought to involve early-onset neurovascular abnormalities. Hitherto studies on AD-associated neurovascular injury have used animal models that exhibit only a subset of AD-like pathologies and demonstrated some Aβ-dependent vascular dysfunction and destabilization of neuronal network. The present work focuses on the early stage of disease progression and uses TgF344-AD rats that recapitulate a broader repertoire of AD-like pathologies to investigate the cerebrovascular and neuronal network functioning using in situ two-photon fluorescence microscopy and laminar array recordings of local field potentials, followed by pathological analyses of vascular wall morphology, tau hyperphosphorylation, and amyloid plaques. Concomitant to widespread amyloid deposition and tau hyperphosphorylation, cerebrovascular reactivity was strongly attenuated in cortical penetrating arterioles and venules of TgF344-AD rats in comparison to those in non-transgenic littermates. Blood flow elevation to hypercapnia was abolished in TgF344-AD rats. Concomitantly, the phase-amplitude coupling of the neuronal network was impaired, evidenced by decreased modulation of theta band phase on gamma band amplitude. These results demonstrate significant neurovascular network dysfunction at an early stage of AD-like pathology. Our study identifies early markers of pathology progression and call for development of combinatorial treatment plans.

Wide field fluorescent imaging of extracellular spatiotemporal potassium dynamics in vivo

Potassium homeostasis is fundamental for the physiological functioning of the brain. Increased [K(+)] in the extracellular fluid has a major impact on neuronal physiology and can lead to ictal events. Compromised regulation of extracellular [K(+)] is involved in generation of seizures in animal models and potentially also in humans. For this reason, the investigation of K(+) spatio-temporal dynamics is of fundamental importance for neuroscientists in the field of epilepsy and other related pathologies. To date, the majority of studies investigating changes in extracellular K(+) have been conducted using a micropipette filled with a K(+) sensitive solution. However, this approach presents a major limitation: the area of the measurement is circumscribed to the tip of the pipette and it is not possible to know the spatiotemporal distribution or origin of the focally measured K(+) signal. Here we propose a novel approach, based on wide field fluorescence, to measure extracellular K(+) dynamics in neural tissue. Recording the local field potential from the somatosensory cortex of the mouse, we compared responses obtained from a K(+)-sensitive microelectrode to the spatiotemporal increases in fluorescence of the fluorophore, Asante Potassium Green-2, in physiological conditions and during 4-AP induced ictal activity. We conclude that wide field imaging is a valuable and versatile tool to measure K(+) dynamics over a large area of the cerebral cortex and is capable of capturing fast dynamics such as during ictal events. Moreover, the present technique is potentially adaptable to address questions regarding spatiotemporal dynamics of other ionic species.

Astrocytic gap junction blockade markedly increases extracellular potassium without causing seizures in the mouse neocortex.

Extracellular potassium concentration, [K+]o, is a major determinant of neuronal excitability. In the healthy brain, [K+]o levels are tightly controlled. During seizures, [K+]o increases up to 15mM and is thought to cause seizures due to its depolarizing effect. Although astrocytes have been suggested to play a key role in the redistribution (or spatial buffering) of excess K+ through Connexin-43 (Cx43)-based Gap Junctions (GJs), the relation between this dynamic regulatory process and seizure generation remains unknown. Here we contrasted the role of astrocytic GJs and hemichannels by studying the effect of GJ and hemichannel blockers on [K+]o regulation in vivo. [K+]o was measured by K+-sensitive microelectrodes. Neuronal excitability was estimated by local field potential (LFP) responses to forepaw stimulation and changes in the power of resting state activity. Starting at the baseline [K+]o level of 1.61±0.3mM, cortical microinjection of CBX, a broad spectrum connexin channel blocker, increased [K+]o to 11±3mM, Cx43 GJ/hemichannel blocker Gap27 increased it from 1.9±0.7 to 9±1mM. At these [K+]o levels, no seizures were observed. Cx43 hemichannel blockade with TAT-Gap19 increased [K+]o by only ~1mM. Microinjection of 4-aminopyridine, a known convulsant, increased [K+]o to ~10mM and induced spontaneously recurring seizures, whereas direct application of K+ did not trigger seizure activity. These findings are the first in vivo demonstration that astrocytic GJs are major determinants for the spatial buffering of [K+]o and that an increase in [K+]o alone does not trigger seizures in the neocortex.

Hungry Neurons: Metabolic Insights on Seizure Dynamics

Epilepsy afflicts up to 1.6% of the population and the mechanisms underlying the appearance of seizures are still not understood. In past years, many efforts have been spent trying to understand the mechanisms underlying the excessive and synchronous firing of neurons. Traditionally, attention was pointed towards synaptic (dys)function and extracellular ionic species (dys)regulation. Recently, novel clinical and preclinical studies explored the role of brain metabolism (i.e., glucose utilization) of seizures pathophysiology revealing (in most cases) reduced metabolism in the inter-ictal period and increased metabolism in the seconds preceding and during the appearance of seizures. In the present review, we summarize the clinical and preclinical observations showing metabolic dysregulation during epileptogenesis, seizure initiation, and termination, and in the inter-ictal period. Recent preclinical studies have shown that 2-Deoxyglucose (2-DG, a glycolysis blocker) is a novel therapeutic approach to reduce seizures. Furthermore, we present initial evidence for the effectiveness of 2-DG in arresting 4-Aminopyridine induced neocortical seizures in vivo in the mouse.

Neurovascular unit remodelling in the subacute stage of stroke recovery

Abstract Brain plasticity following focal cerebral ischaemia has been observed in both stroke survivors and in preclinical models of stroke. Endogenous neurovascular adaptation is at present incompletely understood yet its potentiation may improve long‐term functional outcome. We employed longitudinal MRI, intracranial array electrophysiology, Montoya Staircase testing, and immunofluorescence to examine function of brain vessels, neurons, and glia in addition to forelimb skilled reaching during the subacute stage of ischemic injury progression. Focal ischemic stroke (˜100 mm3 or ˜20% of the total brain volume) was induced in adult Sprague‐Dawley rats via direct injection of endothelin‐1 (ET‐1) into the right sensori‐motor cortex, producing sustained impairment in left forelimb reaching ability. Resting perfusion and vascular reactivity to hypercapnia in the peri‐lesional cortex were elevated by approximately 60% and 80% respectively seven days following stroke. At the same time, the normal topological pattern of local field potential (LFP) responses to peripheral somatosensory stimulation was abolished and the average power of spontaneous LFP activity attenuated by approximately 50% relative to the contra‐lesional cortex, suggesting initial response attenuation within the peri‐infarct zone. By 21 days after stroke, perilesional blood flow resolved, but peri‐lesional vascular reactivity remained elevated. Concomitantly, the LFP response amplitudes increased with distance from the site of ET‐1 injection, suggesting functional remodelling from the core of the lesion to its periphery. This notion was further buttressed by the lateralization of spontaneous neuronal activity: by day 21, the average ipsi‐lesional power of spontaneous LFP activity was almost twice that of the contra‐lesional cortex. Over the observation period, the peri‐lesional cortex exhibited increased vascular density, along with neuronal loss, astrocytic activation, and recruitment and activation of microglia and macrophages, with neuronal loss and inflammation extending beyond the peri‐lesional cortex. These findings highlight the complex relationship between neurophysiological state and behaviour and provide evidence of highly dynamic functional changes in the peri‐infarct zone weeks following the ischemic insult, suggesting an extended temporal window for therapeutic interventions.

Early stage attenuation of phase amplitude coupling in the hippocampus and medial prefrontal cortex in a transgenic rat model of AD

Alzheimers disease (AD) is pathologically characterized by amyloid‐β peptide (Aβ) accumulation, neurofibrillary tangle formation, and neurodegeneration. Preclinical studies on neuronal impairments associated with progressive amyloidosis have demonstrated some Aβ‐dependent neuronal dysfunction including modulation of gamma‐aminobutyric acid‐ergic signaling. The present work focuses on the early stage of disease progression and uses TgF344‐AD rats that recapitulate a broad repertoire of AD‐like pathologies to investigate the neuronal network functioning using simultaneous intracranial recordings from the hippocampus (HPC) and the medial prefrontal cortex (mPFC), followed by pathological analyses of gamma‐aminobutyric acid (GABAA) receptor subunits α1, α5, and δ, and glutamic acid decarboxylases (GAD65 and GAD67). Concomitant to amyloid deposition and tau hyperphosphorylation, low‐gamma band power was strongly attenuated in the HPC and mPFC of TgF344‐AD rats in comparison to those in non‐transgenic littermates. In addition, the phase‐amplitude coupling of the neuronal networks in both areas was impaired, evidenced by decreased modulation of theta band phase on gamma band amplitude in TgF344‐AD animals. Finally, the gamma coherence between HPC and mPFC was attenuated as well. These results demonstrate significant neuronal network dysfunction at an early stage of AD‐like pathology. This network dysfunction precedes the onset of cognitive deficits and is likely driven by Aβ and tau pathologies.

Neurogliovascular dysfunction in a model of repeated traumatic brain injury

Traumatic brain injury (TBI) research has focused on moderate to severe injuries as their outcomes are significantly worse than those of a mild TBI (mTBI). However, recent epidemiological evidence has indicated that a series of even mild TBIs greatly increases the risk of neurodegenerative and psychiatric disorders. Neuropathological studies of repeated TBI have identified changes in neuronal ionic concentrations, axonal injury, and cytoskeletal damage as important determinants of later life neurological and mood compromise; yet, there is a paucity of data on the contribution of neurogliovascular dysfunction to the progression of repeated TBI and alterations of brain function in the intervening period. Methods: Here, we established a mouse model of repeated TBI induced via three electromagnetically actuated impacts delivered to the intact skull at three-day intervals and determined the long-term deficits in neurogliovascular functioning in Thy1-ChR2 mice. Two weeks post the third impact, cerebral blood flow and cerebrovascular reactivity were measured with arterial spin labelling magnetic resonance imaging. Neuronal function was investigated through bilateral intracranial electrophysiological responses to optogenetic photostimulation. Vascular density of the site of impacts was measured with in vivo two photon fluorescence microscopy. Pathological analysis of neuronal survival and astrogliosis was performed via NeuN and GFAP immunofluorescence. Results: Cerebral blood flow and cerebrovascular reactivity were decreased by 50±16% and 70±20%, respectively, in the TBI cohort relative to sham-treated animals. Concomitantly, electrophysiological recordings revealed a 97±1% attenuation in peri-contusional neuronal reactivity relative to sham. Peri-contusional vascular volume was increased by 33±2% relative to sham-treated mice. Pathological analysis of the peri-contusional cortex demonstrated astrogliosis, but no changes in neuronal survival. Conclusion: This work provides the first in-situ characterization of the long-term deficits of the neurogliovascular unit following repeated TBI. The findings will help guide the development of diagnostic markers as well as therapeutics targeting neurogliovascular dysfunction.

Oophorectomy Reduces Estradiol Levels and Long-Term Spontaneous Neurovascular Recovery in a Female Rat Model of Focal Ischemic Stroke

Although epidemiological evidence suggests significant sex and gender-based differences in stroke risk and recovery, females have been widely under-represented in preclinical stroke research. The neurovascular sequelae of brain ischemia in females, in particular, are largely uncertain. We set out to address this gap by a multimodal in vivo study of neurovascular recovery from endothelin-1 model of cortical focal-stroke in sham vs. ovariectomized female rats. Three weeks post ischemic insult, sham operated females recapitulated the phenotype previously reported in male rats in this model, of normalized resting perfusion but sustained peri-lesional cerebrovascular hyperreactivity. In contrast, ovariectomized (Ovx) females showed reduced peri-lesional resting blood flow, and elevated cerebrovascular responsivity to hypercapnia in the peri-lesional and contra-lateral cortices. Electrophysiological recordings showed an attenuation of theta to low-gamma phase-amplitude coupling in the peri-lesional tissue of Ovx animals, despite relative preservation of neuronal power. Further, this chronic stage neuronal network dysfunction was inversely correlated with serum estradiol concentration. Our pioneering data demonstrate dramatic differences in spontaneous recovery in the neurovascular unit between Ovx and Sham females in the chronic stage of stroke, underscoring the importance of considering hormonal-dependent aspects of the ischemic sequelae in the development of novel therapeutic approaches and patient recruitment in clinical trials.

TRANSIENT HYPERTENSION-INDUCED CEREBROVASCULAR STRUCTURAL AND FUNCTIONAL DEFICITS IN TGF344 AD RATS ARE RESCUED BY COMBINATION TREATMENT

Background:Midlife hypertension is a major risk factors for lateonset AD, yet restoration of normotension has very modest effects on risk of dementia. This study aimed to characterize the molecular and pathophysiological mechanisms by which transient hypertension contributes to dementia. Furthermore, our combined therapeutic approach was designed to target AD pathophysiology in combination with vascular injury. Methods:We modeled transient midlife hypertension by administration of L-NAME, a nitric oxide synthase inhibitor, followed by restoration of normotension by cessation of L-NAME administration. A combination of twophoton fluorescence microscopy, biochemistry and immunohistochemistry was used to examine cerebrovascular structure and function. Following return to normotension, scyllo-inositol (SI) treatment was initiated prior to human umbilical cord perivascular cell (HUCPVC) transplantation. Continuous ASL MRI was performed post-L-NAME treatment, and at endpoint to assess the effects of combination treatment on cerebrovascular function. Results: TgAD and nTg rats developed moderate hypertension that returned to normotension 4-weeks post cessation of LNAME. Cerebrovascular damage was greater and sustained in the hypertensive vs. vehicle administered rats. The combined treatment, HUCPVC + SI, rescued the cerebrovascular deficits induced by transient hypertension to a greater extent than either treatment in isolation. We found that in TgAD rats, L-NAME treatment increased cerebrovascular wall hardening and increased deposition of both parenchymal and vascular amyloid; combination treatment reduced only cerebrovascular amyloid Conclusions: Transient hypertension causes sustained cerebrovascular damage in a transgenic model of Alzheimer’s disease and thus multiple drug treatment paradigms may be necessary to address AD with comorbid vascular disease.

Modulation of the peri‐infarct neurogliovascular function by delayed COX‐1 inhibition

Stroke is the leading cause of adult disability worldwide. The absence of more effective interventions in the chronic stage—that most patients stand to benefit from—reflects uncertainty surrounding mechanisms that govern recovery. The present work investigated the effects of a novel treatment (selective cyclooxygenase‐1, COX‐1, inhibition) in a model of focal ischemia.