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Type 2 diabetes is associated with diabetic cognopathy. Anti-hyperglycemic sodium glucose transporter 2 (SGLT2) inhibitors have shown promise in reducing cognitive impairment in mice with type 2 diabetes mellitus. We recently described marked ultrastructural (US) remodeling of the neurovascular unit (NVU) in type 2 diabetic db/db female mice. Herein, we tested whether the SGLT-2 inhibitor, empagliflozin (EMPA), protects the NVU from abnormal remodeling in cortical gray and subcortical white matter. Ten-week-old female wild-type and db/db mice were divided into lean controls (CKC, n = 3), untreated db/db (DBC, n = 3), and EMPA-treated db/db (DBE, n = 3). Empagliflozin was added to mouse chow to deliver 10 mg kg−1 day−1 and fed for ten weeks, initiated at 10 weeks of age. Brains from 20-week-old mice were immediately immersion fixed for transmission electron microscopic study. Compared to CKC, DBC exhibited US abnormalities characterized by mural endothelial cell tight and adherens junction attenuation and/or loss, pericyte attenuation and/or loss, basement membrane thickening, glia astrocyte activation with detachment and retraction from mural cells, microglia cell activation with aberrant mitochondria, and oligodendrocyte–myelin splitting, disarray, and axonal collapse. We conclude that these abnormalities in the NVU were prevented in DBE. Empagliflozin may provide neuroprotection in the diabetic brain.

The transmission cycle of chikungunya virus (CHIKV) requires that mosquito vectors get persistently infected with the virus, following its oral acqsuisition from a vertebrate host. The mosquito midgut is the initial organ that gets infected with orally acquired CHIKV. Following its replication in the midgut epithelium, the virus exits the midgut and infects secondary tissues including the salivary glands before being transmitted to another host. Here, we investigate the pattern of CHIKV dissemination from the midgut of Aedes aegypti at the ultrastructural level. Bloodmeal ingestion caused overstretching of the midgut basal lamina (BL), which was disrupted in areas adjacent to muscles surrounding the midgut as shown by scanning electron microscopy (SEM). Using both transmission electron microscopy (TEM) and focused ion beam scanning electron microscopy (FIB-SEM) to analyze midgut preparations, mature chikungunya (CHIK) virions were found accumulating at the BL and within strands of the BL at 24–32 h post-infectious bloodmeal (pibm). From 48 h pibm onwards, virions no longer congregated at the BL and became dispersed throughout the basal labyrinth of the epithelial cells. Ingestion of a subsequent, non-infectious bloodmeal caused mature virions to congregate again at the midgut BL. Our study suggests that CHIKV needs a single replication cycle in the midgut epithelium before mature virions directly traverse the midgut BL during a relatively narrow time window, within 48 h pibm.

Aedes aegypti is the primary vector of Zika virus (ZIKV), a flavivirus which typically presents itself as febrile-like symptoms in humans but can also cause neurological and pregnancy complications. The transmission cycle of mosquito-borne arboviruses such as ZIKV requires that various key tissues in the female mosquito get productively infected with the virus before the mosquito can transmit the virus to another vertebrate host. Following ingestion of a viremic blood-meal from a vertebrate, ZIKV initially infects the midgut epithelium before exiting the midgut after blood-meal digestion to disseminate to secondary tissues including the salivary glands. Here we investigated whether smaller Ae. aegypti females resulting from food deprivation as larvae exhibited an altered vector competence for blood-meal acquired ZIKV relative to larger mosquitoes. Midguts from small 'Starve' and large 'Control' Ae. aegypti were dissected to visualize by transmission electron microscopy (TEM) the midgut basal lamina (BL) as physical evidence for the midgut escape barrier showing Starve mosquitoes with a significantly thinner midgut BL than Control mosquitoes at two timepoints. ZIKV replication was inhibited in Starve mosquitoes following intrathoracic injection of virus, however, Starve mosquitoes exhibited a significantly higher midgut escape and population dissemination rate at 9 days post-infection (dpi) via blood-meal, with more virus present in saliva and head tissue than Control by 10 dpi and 14 dpi, respectively. These results indicate that Ae. aegypti developing under stressful conditions potentially exhibit higher midgut infection and dissemination rates for ZIKV as adults, Thus, variation in food intake as larvae is potentially a source for variable vector competence levels of the emerged adults for the virus.

In the mosquito, the midgut epithelium is the initial tissue to become infected with an arthropod-borne virus (arbovirus) that has been acquired from a vertebrate host along with a viremic bloodmeal. Following its replication in midgut epithelial cells, the virus needs to exit the midgut and infect secondary tissues including the salivary glands before it can be transmitted to another vertebrate host. The viral exit mechanism from the midgut, the midgut escape barrier (MEB), is poorly understood although it is an important determinant of mosquito vector competence for arboviruses. Using chikungunya virus (CHIKV) as a model in Aedes aegypti, we demonstrate that the basal lamina (BL) of the extracellular matrix (ECM) surrounding the midgut constitutes a potential barrier for the virus. The BL, predominantly consisting of collagen IV and laminin, becomes permissive during bloodmeal digestion in the midgut lumen. Bloodmeal digestion, BL permissiveness, and CHIKV dissemination are coincident with increased collagenase activity, diminished collagen IV abundance, and BL shredding in the midgut between 24-32 h post-bloodmeal. This indicates that there may be a window-of-opportunity during which the MEB in Ae. aegypti becomes permissive for CHIKV. Matrix metalloproteinases (MMPs) are the principal extracellular endopeptidases responsible for the degradation/remodeling of the ECM including the BL. We focused on Ae. aegypti (Ae)MMP1, which is expressed in midgut epithelial cells, is inducible upon bloodfeeding, and shows collagenase (gelatinase) activity. However, attempts to inhibit AeMMP activity in general or specifically that of AeMMP1 did not seem to affect its function nor produce an altered midgut escape phenotype. As an alternative, we silenced and overexpressed the Ae. aegypti tissue inhibitor of metalloproteinases (AeTIMP) in the mosquito midgut. AeTIMP was highly upregulated in the midgut during bloodmeal digestion and was able to inhibit MMP activity in vitro. Bloodmeal-inducible, midgut-specific overexpression of AeTIMP or its expression via a recombinant CHIKV significantly increased midgut dissemination rates of the virus. Possibly, AeTIMP overexpression affected BL degradation and/or restoration thereby increasing the midgut dissemination efficiency of the virus.