Empagliflozin Ameliorates Type 2 Diabetes-Induced Ultrastructural Remodeling of the Neurovascular Unit and Neuroglia in the Female db/ db Mouse

Abstract

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.

Keywords: astrocyte; endothelial cell; microglia; mitochondria; myelin; neuroglia; oligodendrocyte; pericyte; sodium glucose co-transporter 2 inhibitor (SGLT2i); white matter.

Conflict of interest statement

Unrestricted research support from Boehringer Ingelheim was provided to V.G.D. All other authors declare no conflict of interest.

Figures

Figure 1

The neurovascular unit (NVU) in lean control (panels (A–C): CKC), untreated diabetic (panel (D): DBC), and diabetic mice treated with empagliflozin (EMPA) (panel (E): DBE). Panels (A) and (B) illustrate the normal ultrastructural morphology of the (NVU) in control CKC at higher magnification in order to demonstrate with greater clarity the contents of each cell comprising the NVU. Magnification 4000×; scale bar = 1 µm. Modified with permission from Reference [1]. Panel (C) illustrates a probing ramified microglia cell (rMGC) (pseudo-colored green) probing the NVU with an intact (pseudo-colored golden) halo or corona of astrocytes (ACs) within the confines of the neuropil. Panel (D) depicts an invasive (pseudo-colored red) activated microglia cell (aMGC) that has completely engulfed the NVU (uncolored) with markedly thickened basement membranes in DBC. Also, note the increased electron density and volume of chromatin within the aMGC nuclei in this image in addition to the detachment and retraction of ACs of the NVU. It is also important to note that there is a loss of pericytes and the normal intact ACs to form the halo—corona as in CKC and DBE (panels (C) and (E)). Panel (E) depicts an intact NVU, which is in close contact to two adjacent pyramidal (Pry) neurons in DBE and note the intact (pseudo-colored golden) AC halo—corona enveloping the endothelial cells similar to CKC controls with intact tight and adherens junctions within the endothelial cells (ECs) (arrows). Magnification 800×; scale bar = 2 µm (panels (C–E)). Figure images throughout text are color-coded with control CKC images outlined in green; diabetic db/db DBC in red; EMPA (SGLT2 inhibitor) treated DBE in blue in order to readily assist the reader in identification of each cohort. Cap = capillary; CL = capillary lumen.

Figure 2

Photomicrograph of brain specimen for transmission electron microscopic studies. The left hemisphere was utilized for this study. Cortical gray matter tissue specimens were obtained just cephalad to the mid-cortex dashed line (asterisk). Scale = 0.1 cm. Brain measured approximately 1.4 × 1.5 cm upon removal.

Figure 3

Empagliflozin protects the NVU from basement membrane (BM) thickening, loss of blood–brain barrier endothelial tight and adherens junctions and pericytes. Panel (A) depicts the normal ultrastructure morphologic phenotype of the NVU in the CKC. Panels (B) and (C) depict thickening of the NVU basement membrane, activation of NVU endothelial cells (white blood cell (WBC)—lymphocyte adhesion) with loss of blood–brain barrier tight and adherens junctions, inflammation and activation of endothelial cell in panel (B), and detachment and retraction of astrocytes (AC—hashtags and red pseudo-colored astrocytes) in panels (B) and (C). Panels (D), (E), and (F) depict the intact NVUs with protection from NVU cellular remodeling in DBE, and note they are similar to CKC (panel (A)). Note the intact TJ/AJ in panel (D) (arrows), the intact pericyte nucleus and pericyte process (PcP) in panel (E), and the intact astrocytes in panels (D), (E), and (F). At this magnification it is difficult to visualize the endothelial cell(s) that form the inner lining of the NVU capillary lumen (CL). Magnification 2000×; scale bar = 1 µm. AC = astrocyte end feet; aMGC = activated amoeboid microglial cell; rMGC = ramified microglial cell; CL = capillary lumen; PcN = pericyte nucleus; PcP = pericyte process.

Figure 4

Basement membrane thickening, activated microglia invasion, and detachment of astrocyte foot processes are protected with empagliflozin. Panel (A) depicts the invasion of the activated MGC (aMGC) to totally engulf the NVU with detachment of AC end-foot processes (pseudo-colored blue; drAC = detached retracted astrocyte). Also, note the aberrant mitochondria (aMt) (pseudo-colored yellow with red outline) in DBC, which may be responsible for reactive oxygen species (ROS) production and leakage. Dashed arrows depict the detachment and retraction of the AC associated with aMGC invasion of the NVU. Panel (B) illustrates how empagliflozin protects the NVU from aberrant remodeling in DBE. Note the intact AC end feet (pseudo-colored golden) and that the mitochondria in DBE are electron dense and not aberrant as in the DBC. Panel (B) is a higher magnification of panel F in Figure 3. Magnification 2500×; scale bar = 1 µm. aMGC = activated microglia cell; aMt = aberrant mitochondria; drAC = detached retracted AC; EC = endothelial cell; CL = NVU capillary lumen; Pc = pericyte foot process; RBC = red blood cell.

Figure 5

Aberrant mitochondria in endothelial cells, pericytes, and foot processes, astrocytes, oligodendrocytes, myelinated and unmyelinated neurons. Panels (A–F) demonstrate that aberrant mitochondria (aMt) were found to be present in multiple cells in addition to activated microglia cells (aMGCs). The aMt are pseudo-colored in each of these panels (yellow outlined in red lines) in order to allow their rapid recognition. Panels depict the aMt characterized by swollen mitochondria (Mt), loss of electron-dense Mt matrix and crista. Panel (A) illustrates the aMt within the endothelial cells and surrounding aMGC. Panel (B) depicts aMt within AC cytoplasm. Panel (C) demonstrates aMt in pericytes and foot processes (Pc and Pcfp). Panel (D) depicts aMt in a dysmyelinated neuronal axon. Panel (E) depicts aMt within an oligodendrocyte cytoplasm and Panel (F) illustrates aMt in an AC to the left and an unmyelinated axon on the right within the neuropil. Scale bars = 0.5 μm in all images except for panel (B) which has a scale bar = 1 μm [2].

Figure 6

Diabetes-induced aberrant mitochondria remodeling was prevented by empagliflozin. Note that in all panels (A–F) the mitochondria are of smaller size and that the mitochondria matrix is electron dense (pseudo-colored green encircled with white lines) in contrast to the swollen aMt with loss of mitochondrial matrix electron density and loss of crista in the previous Figure 5. Magnification 6000× (panel (A)); 2000× (panel (B)), and 4000× (panels (C–F)); Scale bars = 0.5 μm in all images except for panel (B) which has a scale bar = 1 μm. AC = astrocyte; EC = endothelial cell; M = myelin; OL= oligodendrocyte; PcP = pericyte foot process; UnM = unmyelinated.

Figure 7

Cortical gray matter myelin remodeling was protected with empagliflozin treatment. Panels (A) and (B) demonstrate normal myelination and electron-dense mitochondria (Mt) in the cortical gray matter. Panels (C) and (D) depict marked myelin splitting and separation in DBC, and also note the aberrant mitochondria (aMt) within the axons (pseudo-colored yellow encircled by red outlines) as compared to the normal axonal mitochondria in CKC and DBC (pseudo-colored green with white outlines). Dysmyelination and aMt remodeling were prevented with empagliflozin treatment (panels (E), (F)). Magnification 4000×; scale bar = 0.5 µm.

Figure 8

Diabetes-induced myelin disarray in the transitional zone was protected by empagliflozin treatment. Panel (A) illustrates the findings in the CKC. Panels (B) and (C) depict the marked myelin disarray, and note the more normal appearing myelin in panel (B) with the demarcation line (yellow dashed line) between more normal myelin and the myelin disarray. Also note the open arrows in panels (B) and (C), which depict axonal collapse within the myelinated axons. Panel (C) demonstrates an oligodendrocyte (OL) with increased chromatin condensation. In contrast note how treatment with empagliflozin results in less myelin disarray and illustrates normal oligodendrocyte morphology without chromatin condensation within the nucleus. Magnification 2000× in panels (A) and (D) with scale bar =1 µm. Magnification 1200×; scale bar = 1–2 µm (panels (B), (C), (E), (F)).

Figure 9

Diabetes-induced axonal collapse was prevented by empagliflozin. Panel (A) illustrates the normal morphology of myelinated axons and note the compactness of the myelin lamella. Panel (B) depicts multiple myelinated axons with collapsed axoplasm of axons (AX) (red double arrows) as compared to the myelinated neurons in control CKC (panel (A)). Panel (C) demonstrates that empagliflozin treatment protects axons from collapsing and appears similar to CKC in panel (A). Magnification 4000×; Bar = 0.5 µm. Ax = axon.

Figure 10

Diabetes-induced transitional zone subcortical white matter myelin and axonal remodeling was prevented with empagliflozin. Panels (A) and (C) demonstrate normal axon and myelin ultrastructural morphology in the transitional zone. Panels (B) and (D) depict myelin splitting, disarray, and axonal collapse, respectively (arrows and double arrows). Empagliflozin prevented the aberrant myelin and axonal remodeling (panels (E), (F)). Insert in panel (F) depicts the intact myelin lamellar units. Magnifications 2000×; scale bar = 1 µm (panels (A), (B)); magnifications 4000×; scale bar = 0.5 µm (panels (C)–(F)). Magnification 20,000×; scale bar = 100 nm (insert panel (F)). Ax = axon; TZ SCWM = transitional zone subcortical white matter.

Figure 11

Fixed images of the focused ion-beam (FIB)/SEM emphasizing ultrastructural normal myelin in relation to the surrounding neuropil in DBE. Panel (A) depicts the markedly electron-dense myelin (M), and note that the mitochondria (Mt) are electron dense without any abnormal remodeling changes of aberrant Mt as found in DBC in prior images (especially Figure 3 and Figure 4). This 2D type of image with left and right sides folded up at the regions of the black-dashed lines illustrates the marked electron density of myelin allowing the computer to stack and create 3D images in panel (B). Note the large pyramidal (PYR) cell with electron-dense, normal appearing cytoplasmic mitochondria in both panels (A) and (B) within the cytoplasm of PYR cells. Importantly, there is no splitting, separation or disarray as noted in previously-fixed ultrastructural DBC images. Panel (B) illustrates the computerized developed 3D-type image of myelin as a result of stacking electron dense myelin, which allowed the computer program to pseudo-color myelin golden. Note the uniformity of the golden myelin without separation, splitting or disarray as compared to the previous DBC figure images.

Figure 12

Labeled pre-run Supplementary Materials Video S1 fixed images comparing DBC and DBE. Panel (A) depicts the multiple enlarged more electron lucent aberrant mitochondria with loss of electron-dense mitochondria matrix and crista (arrows) in the diabetic DBC db/db model. In the empagliflozin-treated DBE model (panel (B)), note that the mitochondria are electron dense and smaller (arrows), which are morphologically similar in control non-diabetic lean models as in CKC in previously-fixed images (Figure 1A–C; Figure 3A–C; Figure 7A,B; Figure 8A; Figure 9A; Figure 10A,C) and that the myelinated axons are not split and separated as in previously depicted images and (Video S1). Our observations support that the treatment with empagliflozin protects not only the aberrant Mt but also abnormal myelin remodeling in DBE models. Scale bar = 5 µm. aMGC = activated microglia cell; Lys = lysosome; M = myelin; MGC = microglia cell; N = nucleus; PYR = pyramidal neuron cell.

Figure 13

Stacked fixed images of diabetic DBC (outlined red) and EMPA-treated DBE models (outlined blue). The front pre-run images are labeled in Figure 12. The images in this figure are to demonstrate how Video S1 was created by utilizing approximately 250 stacked sliced images every 20 nm in order to create the Supplementary Materials DBC [2] and DBE video (Video S1). Scale bar = 5 µm.

Figure 14

Possible mechanisms for remodeling changes in diabetic db/db mice and protection by EMPA. We propose an overarching hypothesis that hyperglycemia and glucotoxicity serve as a central injury mechanism to cells and tissues and are definitely upstream of diabetic end-organ complications, which may include microvascular disease and ultrastructure remodeling of the neurovascular unit in the brains of type 2 diabetes mellitus (T2DM) db/db mice and human patients. We have previously identified the observational abnormal remodeling associated with the NVU and its supportive cellular constituents (endothelial cell, pericyte, astrocyte, microglia and regional neurons) [1,2,3]. It is important to note that 10-week administration of EMPA protected the NVU and it supportive cells and neurons from US remodeling. In support of this, a recent paper showed that EMPA administration was associated with decreased cerebral-oxidative stress, increase brain-derived neurotrophic factor, and significantly prevented the impairment of cognitive function [8]. Importantly, note that empagliflozin in DBE models improves glucotoxicity and may also improve insulin and leptin sensitivity. AC = astrocyte; EC = endothelial cell; AGE = advance glycation end product; aMt = aberrant mitochondria; MGC = microglia cell; MMP = matrix metalloproteinase; NADPH Ox = nicotinamide adenine dinucleotide phosphate oxidase; NOX1,2,4 family genes encoding NADPH Ox 1,3,4 Ox = oxidase; PKC = protein kinase C; RAGE = receptor for AGE; RAS = renin angiotensin aldosterone system; RNS = reactive nitrogen species; ROS = reactive oxygen species; TJ/AJ = tight junction/adherens junction.