Ultrastructural Remodeling of the Neurovascular Unit in the Female Diabetic db/db Model–Part II: Microglia and Mitochondria

Obesity, insulin resistance, and type 2 diabetes mellitus are associated with diabetic cognopathy. This study tested the hypothesis that neurovascular unit(s) (NVU) within cerebral cortical gray matter regions may depict abnormal cellular remodeling. The monogenic (Leprdb) female diabetic db/db [BKS.CgDock7m +/+Leprdb/J] (DBC) mouse model was utilized for this ultrastructural study. Upon sacrifice (20 weeks), left-brain hemispheres of the DBC and age-matched nondiabetic control C57BL/KsJ (CKC) mice were immediately immersion-fixed. We observed an attenuation/loss of endothelial blood–brain barrier tight/adherens junctions and pericytes, thickened basement membranes, adherent red and white blood cells, neurovascular unit microbleeds and pathologic remodeling of protoplasmic astrocytes. In this second of a three-part series, we focus on the observational ultrastructural remodeling of microglia and mitochondria in relation to the NVU in leptin receptor deficient DBC models. This study identified novel ultrastructural core signature remodeling changes, which consisted of invasive activated microglia, microglial aberrant mitochondria with nuclear chromatin condensation and adhesion of white blood cells to an activated endothelium of the NVU. In conclusion, the results implicate activated microglia in NVU uncoupling and the resulting ischemic neuronal and synaptic damage, which may be related to impaired cognition and diabetic cognopathy.


Introduction
Previously, we have overviewed the background, and documented the observations of the 20-week old female db/db [BKS.CgDock7 m +/+Lepr db /J] (DBC) and its comparison to the age-matched control (CKC) model with a focus on ultrastructure protoplasmic astrocyte remodeling in relation to the neurovascular unit (NVU) [1]. We demonstrated marked multicellular ultrastructure remodeling comprised by the following: (i) attenuation and/or loss of blood-brain barrier tight and adherens junctions (TJ/AJ); (iia) adherent red blood cells to endothelial cell(s) (EC) of the NVU; (iib) microbleeds of the NVU; (iic) endothelial cell thinning and activation with white blood cell adherence to  Table 1 [1]. (A) Depicts an astrocyte (AC) with electron lucent cytoplasm with a neuronal pyramidal (PYR) cell immediately adjacent. (B) Depicts a ramified microglial cell (rMGC) with its highly electron-dense cytoplasm and nucleus outlined by white dashed line. (C) Depicts just a partial image of an oligodendrocyte (OL) in the transition zone between the cortical grey matter and white matter. Note the ramified cytoplasmic extensions. Microglia are the smallest of the glia cells and their cytoplasm is the most electron-dense of the neurovascular unit (NVU) cells and brain cortical grey matter. In their nonactivated phenotypic state, they have elongated cytoplasmic process in ramified form. They have an extensive endoplasmic reticulum, Golgi body system and contain multiple mitochondria. Their cytoplasmic processes are known to be capable of extending and contracting. They have a unique morphology of their nuclei with an outer stippled chromatin at its neurolemma and a more stippled diffuse chromatin electrondense appearance of the central nuclei. Magnification 1200×; bar = 2 μm.
Microglial cells are the resident innate immune cells of the brain, which represent approximately 5-20% of the brain neuroglial population and have a large number of membranous and intracellular microglial markers; a large number of signaling molecules, which include numerous microglial cytokines and chemokines [2]. Microglia contribute to the regulation of brain development, shaping synaptic connectivity within neuronal networks and are of major importance in brain defense injury [2][3][4]. In the healthy brain, ramified microglia are constantly surveilling their regional environment and provide the necessary housekeeping-cleaning-gatekeeping functions to maintain brain tissue homeostasis ( Figure S1, Figure S2 and Video S1). Microglia are capable of producing numerous free radicals (superoxide, reduced nicotinamide adenine dinucleotide phosphate (NADPH Ox), inducible nitric oxide and mitochondrial-derived reactive oxygen/nitrogen (mtROS)) and are the major killing and phagocytic cell in the brain if bacterial, viral or parasitic infections become invasive. Importantly, microglia are able to return to return to their surveilling-ramified phenotype once the invaders or danger-damage signals have been eradicated and assume their normal cellular debris housekeeping role [2][3][4][5][6][7][8][9][10][11][12].
Mesoderm (yolk sac)-derived microglia cells are the first and main form of active immune defense in the brain [2][3][4][5][6][7][8][9][10][11][12]. Microglia are unique from bone marrow derived peripheral monocytemacrophage cells in that they are not dependent on recruitment from the peripheral systemic circulation but are capable of undergoing proliferation mechanisms if needed [13]. Microglia may play both a protective role of surveillance for injury to the NVU unit ( Figure S2, Figure S3 and Video S2) as well as a possible damaging role to the NVU in the DBC models due to microglia invasiveness Figure 1. Comparison of ramified microglia to glia and pyramidal cells in cortical grey matter layer III to supplement the extracted description from Table 1 [1]. (A) Depicts an astrocyte (AC) with electron lucent cytoplasm with a neuronal pyramidal (PYR) cell immediately adjacent. (B) Depicts a ramified microglial cell (rMGC) with its highly electron-dense cytoplasm and nucleus outlined by white dashed line. (C) Depicts just a partial image of an oligodendrocyte (OL) in the transition zone between the cortical grey matter and white matter. Note the ramified cytoplasmic extensions. Microglia are the smallest of the glia cells and their cytoplasm is the most electron-dense of the neurovascular unit (NVU) cells and brain cortical grey matter. In their nonactivated phenotypic state, they have elongated cytoplasmic process in ramified form. They have an extensive endoplasmic reticulum, Golgi body system and contain multiple mitochondria. Their cytoplasmic processes are known to be capable of extending and contracting. They have a unique morphology of their nuclei with an outer stippled chromatin at its neurolemma and a more stippled diffuse chromatin electron-dense appearance of the central nuclei. Magnification 1200×; bar = 2 µm.
Microglial cells are the resident innate immune cells of the brain, which represent approximately 5-20% of the brain neuroglial population and have a large number of membranous and intracellular microglial markers; a large number of signaling molecules, which include numerous microglial cytokines and chemokines [2]. Microglia contribute to the regulation of brain development, shaping synaptic connectivity within neuronal networks and are of major importance in brain defense injury [2][3][4]. In the healthy brain, ramified microglia are constantly surveilling their regional environment and provide the necessary housekeeping-cleaning-gatekeeping functions to maintain brain tissue homeostasis ( Figure S1, Figure S2 and Video S1). Microglia are capable of producing numerous free radicals (superoxide, reduced nicotinamide adenine dinucleotide phosphate (NADPH Ox), inducible nitric oxide and mitochondrial-derived reactive oxygen/nitrogen (mtROS)) and are the major killing and phagocytic cell in the brain if bacterial, viral or parasitic infections become invasive. Importantly, microglia are able to return to return to their surveilling-ramified phenotype once the invaders or danger-damage signals have been eradicated and assume their normal cellular debris housekeeping role [2][3][4][5][6][7][8][9][10][11][12].
Mesoderm (yolk sac)-derived microglia cells are the first and main form of active immune defense in the brain [2][3][4][5][6][7][8][9][10][11][12]. Microglia are unique from bone marrow derived peripheral monocyte-macrophage cells in that they are not dependent on recruitment from the peripheral systemic circulation but are capable of undergoing proliferation mechanisms if needed [13]. Microglia may play both a protective role of surveillance for injury to the NVU unit ( Figure S2, Figure S3 and Video S2) as well as a possible damaging role to the NVU in the DBC models due to microglia invasiveness with resulting detachment and separation of protoplasmic astrocytes from the BM of the endothelial cells and pericytes of the NVU [1].
We originally hypothesized that microglia might demonstrate some of their reactive changes similar to our previous observations in the diet-induced obesity and insulin resistant Western models with impaired intermittent glucose elevation [14].
Microglia in CKC animals display distinct phenotypes in transmission electron microscopy (TEM) (Joel 1400-EX TEM JOEL (JOEL, Peabody, MA, USA) images and these cells will be referred to as ramified microglial cells (rMGC). These cells survey their surrounding milieu of endothelial cells, pericytes, astrocytes of the NVU and neurons. They maintain this ramified phenotype and function in association with intact tight and adherence junctions of the ECs blood-brain barrier and the intact adherent protoplasmic astrocytes to the basement membrane of the NVU. Ramified microglia cells are constantly prepared to undergo a rapid diverse phenotypic remodeling change to what we will reference as the activated-amoeboid microglial cell phenotype (aMGC). These changes may be due to morphological remodeling and/or the expressions of their cell surface receptors in response to danger or damage signals such as pathogen associated molecular patterns (PAMPs) or damage associated molecular patterns (DAMPS) due to oxidized/glycated proteins/polypeptides, lipids, and nucleic acids from their diabetic hyperglycemic microenvironment [2][3][4][5][6][7][8][9][10][11][12][13]. The phenotypic remodeling changes need to be qualified by the model (i.e., control CKC or diabetic DBC), the region of the brain (as in our gray matter cortical layer III), the disease state (obesity, insulin resistance and type 2 diabetes mellitus (T2DM)) and their age (20 weeks) [2][3][4][5][6][7][8][9][10][11][12][13]. Activated microglial phenotypes have been classified by some to be similar to peripheral macrophages, i.e., M1 (classically activated macrophages) and M2 (alternatively activated macrophages) cells [7,8]; however, the possibly more preferred method of identification of MGCs relies on individual cell surface markers or their response to inducible cytokines [9][10][11]. We have chosen to utilize only rMGC or aMGC when referring to our morphofunctional-pathomorphologic phenotypic polarization in TEM observations ( Figure 2). with resulting detachment and separation of protoplasmic astrocytes from the BM of the endothelial cells and pericytes of the NVU [1]. We originally hypothesized that microglia might demonstrate some of their reactive changes similar to our previous observations in the diet-induced obesity and insulin resistant Western models with impaired intermittent glucose elevation [14].
Microglia in CKC animals display distinct phenotypes in transmission electron microscopy (TEM) (Joel 1400-EX TEM JOEL (JOEL, Peabody, MA, USA) images and these cells will be referred to as ramified microglial cells (rMGC). These cells survey their surrounding milieu of endothelial cells, pericytes, astrocytes of the NVU and neurons. They maintain this ramified phenotype and function in association with intact tight and adherence junctions of the ECs blood-brain barrier and the intact adherent protoplasmic astrocytes to the basement membrane of the NVU. Ramified microglia cells are constantly prepared to undergo a rapid diverse phenotypic remodeling change to what we will reference as the activated-amoeboid microglial cell phenotype (aMGC). These changes may be due to morphological remodeling and/or the expressions of their cell surface receptors in response to danger or damage signals such as pathogen associated molecular patterns (PAMPs) or damage associated molecular patterns (DAMPS) due to oxidized/glycated proteins/polypeptides, lipids, and nucleic acids from their diabetic hyperglycemic microenvironment [2][3][4][5][6][7][8][9][10][11][12][13]. The phenotypic remodeling changes need to be qualified by the model (i.e., control CKC or diabetic DBC), the region of the brain (as in our gray matter cortical layer III), the disease state (obesity, insulin resistance and type 2 diabetes mellitus (T2DM)) and their age (20 weeks) [2][3][4][5][6][7][8][9][10][11][12][13]. Activated microglial phenotypes have been classified by some to be similar to peripheral macrophages, i.e., M1 (classically activated macrophages) and M2 (alternatively activated macrophages) cells [7,8]; however, the possibly more preferred method of identification of MGCs relies on individual cell surface markers or their response to inducible cytokines [9][10][11]. We have chosen to utilize only rMGC or aMGC when referring to our morphofunctional-pathomorphologic phenotypic polarization in TEM observations ( Figure 2).

Figure 2.
Microglia spectrum phenotypes in type 2 diabetes mellitus (T2DM). The microglia cell(s) (MGC) are very unique and capable of undergoing a marked diversity of morphological and functional phenotypic remodeling change as they pass along a spectrum of phenotypes from the ramified MGC (rMGC) (green colorization) on the far left-hand side of this cartoon to the chronically activated-amoeboid microglia cells (aMGC) (red colorization) on the right hand in addition to some microglia progressing to a senescent type of MGC on the far right (grey colorization), which may implicate advanced aging. Note the various cytokines profiles associated with each of the rMGC and the aMGC phenotypes. In our age-matched nondiabetic control C57BL/KsJ (CKC) models, the predominate MGC would be rMGC and in the DBC models the predominant microglia would be aMGC since we are only studying the ultrastructural phenotypic ultrastructural remodeling changes. Note that in health or homeostasis we demonstrate that the MGC may be in a flux or spectral change between rMGCs and more activated/amoeboid MGCs. The regions between open arrows may even represent a range of homeostasis between rMGCs and versatile aMGCs phenotypes, while the dashed Figure 2. Microglia spectrum phenotypes in type 2 diabetes mellitus (T2DM). The microglia cell(s) (MGC) are very unique and capable of undergoing a marked diversity of morphological and functional phenotypic remodeling change as they pass along a spectrum of phenotypes from the ramified MGC (rMGC) (green colorization) on the far left-hand side of this cartoon to the chronically activated-amoeboid microglia cells (aMGC) (red colorization) on the right hand in addition to some microglia progressing to a senescent type of MGC on the far right (grey colorization), which may implicate advanced aging. Note the various cytokines profiles associated with each of the rMGC and the aMGC phenotypes. In our age-matched nondiabetic control C57BL/KsJ (CKC) models, the predominate MGC would be rMGC and in the DBC models the predominant microglia would be aMGC since we are only studying the ultrastructural phenotypic ultrastructural remodeling changes. Note that in health or homeostasis we demonstrate that the MGC may be in a flux or spectral change between rMGCs and more activated/amoeboid MGCs. The regions between open arrows may even represent a range of homeostasis between rMGCs and versatile aMGCs phenotypes, while the dashed lines suggest multiple spectral morphologic phenotypes. IL-1β: interleukin 1 beta; IL-14: interleukin 14; IL-10: interleukin 10; TGF-β: Transforming growth factor beta; TNFα: tumor necrosis factor alpha; IL-6: interleukin 6; NADPH Ox: reduced nicotinamide adenine dinucleotide phosphate; Inos: inducible nitric oxide synthase; GSH: glutathione; SOD: superoxide dismutase.

Sample Preparation for Serial Block Face Imaging: Focused Ion Beam/Scanning Electron Microscopy for Supplemental Video 3
Samples were prepared following a modified version of NCMIR (National Center for Microscopy and Imaging Research) methods for three-dimensional (3D) EM. Unless otherwise stated, all reagents wefre purchased from Electron Microscopy Sciences and all specimen preparation was performed at the Electron Microscopy Core Facility, University of Missouri. Tissues were fixed in 2% paraformaldehyde, 2% glutaraldehyde in 100 mM sodium cacodylate buffer pH = 7.35. Next, fixed tissues were rinsed with 100 mM sodium cacodylate buffer, pH = 7.35 containing 130 mM sucrose. Secondary fixation was performed using equal parts 4% osmium tetroxide and 3% potassium ferrocyanide in cacodylate buffer and incubated on ice for 1 h, then rinsed with cacodylate buffer and further with distilled water. En block staining was performed for one hour in a 1% thiocarbohydrazide solution followed by distilled water rinses. Rinsed tissues were incubated in an additional 2% aqueous osmium tetroxide solution for 30 min at room temperature, then rinsed with distilled water. Additional en bloc staining was performed using 1% aqueous uranyl acetate and incubated at 4 • C overnight, then rinsed with distilled water. A final en bloc staining was performed using Walton's Lead Nitrate solution (Lawrence Livermore Laboratory University of California, Livermore, CA, USA) for 30 min at 60 • C. Tissues were rinsed and dehydrated using ethanol, transitioned into acetone, and then infiltrated with Durcapan resin and polymerized at 60 • C overnight. Block faces were prepared using an ultramicrotome (Ultracut UCT, Leica Microsystems, Germany) and a diamond knife (Diatome, Hatfield, PA, USA) and mounted on an SEM stub and coated with 20 nm of platinum using the EMS 150T-ES Sputter Coater (Leica Microsystems Inc. Buffalo Grove, IL USA). Serial block face data was acquired on a Thermo Fisher Scientific Scios Analytical Dualbeam (Hillsboro, OR, USA). The region of interest was identified using established landmarks and protected with a 1-µm layer of platinum using the ion column. Trenches were rough cut to the side and the front of the block face using a high ion beam current (30 kV 5 nA) to expose the desired block face. Next, the block face was polished using an ion beam current of 50 pA prior to collecting serial images using the Slice &View automated software package. Serial sections were cut at a thickness of 20 nm (30 kV 1 nA) and SEM images were acquired at 2 keV and 25 pA using the T1-BSE detector and reverse contrast. Image segmentation was performed using ThermoScientific Amira 6 Software (Thermo Fisher Scientific). Approximately 250 slices were obtained to create the video for Supplementary Video S3.

Microglia Ultrastructure Examination and Observation
Three models per group were studied by TEM (n = 3 in control CKC and in diabetic DBC models). Regions of interest were selected based on the presence of NVU capillaries in gray matter cortical layer III. A total of 60 microglia were eventually studied for all models with 10 from each model providing 30 microglia from CKC and 30 from DBC models. Age-matched nondiabetic control C57BL/KsJ microglia demonstrated 28 of 30 with ramified cytoplasmic projections, 5 of 30 microglia with aMt of 5-6 or more and 3 of 30 with some degree of nuclear chromatin condensation, while in DBC 9 of 30 microglia demonstrated ramifications, 25 of 30 microglia demonstrated aberrant Mt (aMt) with 6-14 aMt and 24 of 30 depicted marked chromatin condensation. Herein, we depict representative ultrastructural remodeling changes in aMGC of the diabetic DBC compared to the rMGC in control CKC models.

Microglial Cell of the Neurovascular Unit in Control CKC Model
Microglial cells reside throughout the brain parenchyma and are frequently adjacent to the endothelial cell, pericytes, mural cells and astrocytes of the NVU in the cortical grey matter of layer III.
The normal ultrastructure morphology of ramified microglial cells in control models (CKC) in relation to the NVU and other interstitial regions are depicted (Figures 3 and 4).

Microglial Cell of the Neurovascular Unit in Control CKC Model
Microglial cells reside throughout the brain parenchyma and are frequently adjacent to the endothelial cell, pericytes, mural cells and astrocytes of the NVU in the cortical grey matter of layer III. The normal ultrastructure morphology of ramified microglial cells in control models (CKC) in relation to the NVU and other interstitial regions are depicted (Figures 3 and 4).

Microglial Cell of the Neurovascular Unit in Control CKC Model
Microglial cells reside throughout the brain parenchyma and are frequently adjacent to the endothelial cell, pericytes, mural cells and astrocytes of the NVU in the cortical grey matter of layer III. The normal ultrastructure morphology of ramified microglial cells in control models (CKC) in relation to the NVU and other interstitial regions are depicted (Figures 3 and 4).   We have previously demonstrated how the ramified microglia with their elongated processes are very responsive and mobile in cleaning Layer III of the cortical grey matter in the diet-induced Western models ( Figure S1, Figure S2 and Video S1). Interestingly, the more proximal somal regions of microglia appear linearly and are observed to be attached to one another similar to box cars of a train between layer III and layer IV and thus, we refer to them as 'trains of microglia' (Video S2). We also observed microglia interrogating the NVU; however, in Western models they appear to back away and do not invade the NVU (as in diabetic DBC models discussed later) since they do not detect any danger or damage signals and do not invade the NVU and appear to back away from the NVU (Video S2).

Microglia Remodeling in Diabetic DBC Models
The aMGC in DBC models were observed to invade the endothelial cells of the NVU and were associated with the detachment and separation / retraction of the astrocyte from the NVU, attenuation and/or loss of the TJ/AJ in the blood brain barrier ( We have previously demonstrated how the ramified microglia with their elongated processes are very responsive and mobile in cleaning Layer III of the cortical grey matter in the diet-induced Western models ( Figure S1, Figure S2 and Video S1). Interestingly, the more proximal somal regions of microglia appear linearly and are observed to be attached to one another similar to box cars of a train between layer III and layer IV and thus, we refer to them as 'trains of microglia' (Video S2). We also observed microglia interrogating the NVU; however, in Western models they appear to back away and do not invade the NVU (as in diabetic DBC models discussed later) since they do not detect any danger or damage signals and do not invade the NVU and appear to back away from the NVU (Video S2).

Microglia Remodeling in Diabetic DBC Models
The aMGC in DBC models were observed to invade the endothelial cells of the NVU and were associated with the detachment and separation / retraction of the astrocyte from the NVU, attenuation and/or loss of the TJ/AJ in the blood brain barrier (BBB) of the NVU and have aberrant mitochondria and chromatin condensation (Figures 5 and 6) Figure 5. Comparison of ramified MGC to an activated MGC in control CKC and diabetic DBC models. (A-D) Depict the ramified (rMGC) (colorized with green nucleus) to demonstrate at low magnification the different morphology of the rMGC as compared to the activated aMGC DBC model (B-D). One notes the aMGC assumes a more amoeboid morphology in the DBC model (B-D) on the right outlined in red as compared to the CKC models on the left with CKC with ramified cytoplasmic extensions (outlined with yellow dashed lines). Also note that the nuclear chromatin is condensed, aggregated/clumped in the DBC models on right when compared to the diffuse nuclear chromatin in the CKC on the left. Importantly, note the major differences between the CKC and DBC in the upper 1-4 numbered major differences between CKC rMGC and DBC aMGC. The low magnification in these images is unable to demonstrate or appreciate the differences in mitochondria in these images. Magnification ×1000 (A-D in CKC) and ×1200 (A-D in DBC); Scale bar = 2 μm. . One notes the aMGC assumes a more amoeboid morphology in the DBC model (B-D) on the right outlined in red as compared to the CKC models on the left with CKC with ramified cytoplasmic extensions (outlined with yellow dashed lines). Also note that the nuclear chromatin is condensed, aggregated/clumped in the DBC models on right when compared to the diffuse nuclear chromatin in the CKC on the left. Importantly, note the major differences between the CKC and DBC in the upper 1-4 numbered major differences between CKC rMGC and DBC aMGC. The low magnification in these images is unable to demonstrate or appreciate the differences in mitochondria in these images. Magnification ×1000 (A-D in CKC) and ×1200 (A-D in DBC); Scale bar = 2 µm.
Microglial cells undergo ultrastructural maladaptive remodeling changes to the cytoplasmic and nuclear components including cytoplasmic aMt that are characterized by markedly swollen mitochondria with loss of electron-dense mitochondrial matrix proteins and crista in the cytoplasm and nuclear chromatin condensation in addition to becoming invasive of the NVU (Figure 7) ( Figure S4 and Video S3).  Microglial cells undergo ultrastructural maladaptive remodeling changes to the cytoplasmic and nuclear components including cytoplasmic aMt that are characterized by markedly swollen mitochondria with loss of electron-dense mitochondrial matrix proteins and crista in the cytoplasm and nuclear chromatin condensation in addition to becoming invasive of the NVU (Figure 7) ( Figure  S4 and Video S3). Activated-amoeboid microglia have a marked increase in cytoplasmic aMt and also demonstrate extensive nuclear chromatin condensation in contrast to a diffuse stippled chromatin pattern in CKC and contain increased numbers of aberrant mitochondria within the cytoplasm, as compared to the ramified microglia in controls Figures 8-12.   Figure 7. Activated microglia become invasive of the neurovascular unit in diabetic DBC models. Panels (C-F) depict how activated MGCs (aMGCs) become invasive to the NVU in DBC models. In contrast to the CKC ramified MGC (rMGC), which are surveilling, aMGC (pseudo-colored red except for the 'frown-faced' aMGC (pseudo-colored blue)) nucleus in (C). Panel F depicts a mononuclear white blood cell (lymphocyte) (WBC) adherent to the luminal NVU endothelial cell. Magnification ×2000 (A); ×1200 (B-D); ×4000 (E); ×3000 (F) with varying scale bars lower left of each panel.
Activated-amoeboid microglia have a marked increase in cytoplasmic aMt and also demonstrate extensive nuclear chromatin condensation in contrast to a diffuse stippled chromatin pattern in CKC and contain increased numbers of aberrant mitochondria within the cytoplasm, as compared to the ramified microglia in controls Figures 8-12.   contrast to the CKC ramified MGC (rMGC), which are surveilling, aMGC (pseudo-colored red except for the 'frown-faced' aMGC (pseudo-colored blue)) nucleus in (C). Panel F depicts a mononuclear white blood cell (lymphocyte) (WBC) adherent to the luminal NVU endothelial cell. Magnification ×2000 (A); ×1200 (B-D); ×4000 (E); ×3000 (F) with varying scale bars lower left of each panel.
Activated-amoeboid microglia have a marked increase in cytoplasmic aMt and also demonstrate extensive nuclear chromatin condensation in contrast to a diffuse stippled chromatin pattern in CKC and contain increased numbers of aberrant mitochondria within the cytoplasm, as compared to the ramified microglia in controls Figures 8-12.  increased in number and demonstrate marked mitochondrial (Mt) swelling with loss of electrondense Mt matrix and crista as compared to control CKC model (Panel A) in diabetic DBC. Aberrant Mt will also be observed in other neurovascular unit cells and neurons in later images. Also, some aMGCs were extremely dark as in panel (B). However, all MGC are dark when compared to other glia and neurons. Magnification ×2000; bar = 1 μm in panels (A,B) and bar = 2 μm in panel (C). CC: chromatin condensation; CL: capillary lumen; N: nucleus. Figure 10. Chromatin condensation in the diabetic DBC. The above images depict ramified and amoeboid-activated microglia in a side-by-side comparison. A ramified microglia (rMGC) on the left with a stippled outer chromatin at the neurolemma and a diffuse stippling of chromatin within the core of the nucleus in the control CKC. In contrast, the amoeboid-activated microglia (aMGC) on the right depicts marked nuclear chromatin condensation in the DBC. Note that in the aMGC of the diabetic DBC the nuclear chromatin condensation-compaction volume is markedly greater than in the nondiabetic control rMGC CKC models. Magnification ×2000; scale bar = 1 μm and was copied and placed within the nuclei to appreciate the sizes of the chromatin.  In contrast, the amoeboid-activated microglia (aMGC) on the right depicts marked nuclear chromatin condensation in the DBC. Note that in the aMGC of the diabetic DBC the nuclear chromatin condensation-compaction volume is markedly greater than in the nondiabetic control rMGC CKC models. Magnification ×2000; scale bar = 1 µm and was copied and placed within the nuclei to appreciate the sizes of the chromatin. mitochondria (aMt) (pseudo-colored yellow with encircling red lines) (arrows) that are markedly increased in number and demonstrate marked mitochondrial (Mt) swelling with loss of electrondense Mt matrix and crista as compared to control CKC model (Panel A) in diabetic DBC. Aberrant Mt will also be observed in other neurovascular unit cells and neurons in later images. Also, some aMGCs were extremely dark as in panel (B). However, all MGC are dark when compared to other glia and neurons. Magnification ×2000; bar = 1 μm in panels (A,B) and bar = 2 μm in panel (C). CC: chromatin condensation; CL: capillary lumen; N: nucleus. Figure 10. Chromatin condensation in the diabetic DBC. The above images depict ramified and amoeboid-activated microglia in a side-by-side comparison. A ramified microglia (rMGC) on the left with a stippled outer chromatin at the neurolemma and a diffuse stippling of chromatin within the core of the nucleus in the control CKC. In contrast, the amoeboid-activated microglia (aMGC) on the right depicts marked nuclear chromatin condensation in the DBC. Note that in the aMGC of the diabetic DBC the nuclear chromatin condensation-compaction volume is markedly greater than in the nondiabetic control rMGC CKC models. Magnification ×2000; scale bar = 1 μm and was copied and placed within the nuclei to appreciate the sizes of the chromatin.   In summary, aMGCs in the diabetic DBC may be characterized by four ultrastructure remodeling core signature changes: (i) Loss of ramified cytoplasmic extensions with amoeboid phenotypes; (ii) invasive phenotypes of the NVU; (iii) aberrant mitochondria with swelling, loss of electron-dense matrix, and loss or fragmented crista; (iv) nuclear chromatin condensation when compared to nondiabetic CKC models.
We have included the newer technology of Focused Ion Beam/Scanning Electron Microscopy (FIB/SEM) videos of the aMGC in the DBC models, which demonstrate not only the nuclear chromatin condensation but also help to better understand the 3D motion of the aM) as they come into and out of view within the cytoplasm of the DBC aMGC ( Figure S4 and Video S3).

Aberrant Mitochondria in the Diabetic DBC Models
Mitochondria play a vital and essential role in nutrient metabolism of brain cells including the vascular mural cells, glia, dendritic synapses and neurons [13,[15][16][17]. Aberrant mitochondria were primarily found to be observed in the cytoplasm of aMGCs; however, these similar aMt remodeling changes also were found in some EC, pericyte (Pc) and Pc foot processes, AC, myelinated and unmyelinated neurons within the neuropil (Figure 13). The observation of aMt in aMGCs may set the stage for redox injuries not only to the microglia itself and their organelles but also the surrounding cells of the NVU.
In summary, aMGCs in the diabetic DBC may be characterized by four ultrastructure remodeling core signature changes: (i) Loss of ramified cytoplasmic extensions with amoeboid phenotypes; (ii) invasive phenotypes of the NVU; (iii) aberrant mitochondria with swelling, loss of electron-dense matrix, and loss or fragmented crista; (iv) nuclear chromatin condensation when compared to nondiabetic CKC models.
We have included the newer technology of Focused Ion Beam/Scanning Electron Microscopy (FIB/SEM) videos of the aMGC in the DBC models, which demonstrate not only the nuclear chromatin condensation but also help to better understand the 3D motion of the aM) as they come into and out of view within the cytoplasm of the DBC aMGC ( Figure S4 and Video S3).

Aberrant Mitochondria in the Diabetic DBC Models
Mitochondria play a vital and essential role in nutrient metabolism of brain cells including the vascular mural cells, glia, dendritic synapses and neurons [13,[15][16][17]. Aberrant mitochondria were primarily found to be observed in the cytoplasm of aMGCs; however, these similar aMt remodeling changes also were found in some EC, pericyte (Pc) and Pc foot processes, AC, myelinated and unmyelinated neurons within the neuropil (Figure 13).

Implications of White Blood Cell (Mononuclear) Lymphocyte Adherence in DBC Models
Adherence of the mononuclear white blood cell (WBC) (lymphocyte) in some NUVs of the diabetic DBC models have allowed not only the concept of an activated endothelium in DBC to be strongly considered as compared to CKC but also have stimulated the question as to the possibility that the aMGC may also be playing a role in signaling the WBC to come in contact with the EC and adhere to an activated endothelium ( Figure 13).
Previously, we had to include images of WBC adherence in Part I because there is such widespread multicellular remodeling that it is difficult to just examine one aspect of cellular remodeling in one image without including other cellular remodeling changes that concurrently occur in DBC models [1]. Also, these glial remodeling changes are associated with hyperglycemia and it is important to understand how hyperglycemia may drive the chronic neuroinflammation and the associated compromise of BBB TJ/AJ, which promote an increase in permeability and leads to memory loss and impaired cognition in T2DM mouse models in the diabetic DBC [18]. Activated microglia are known not only to contain certain cell surface receptors but also are responsible for the secretion of a specialized group of signaling cytokines and chemokines, which include monocyte chemoattractant protein 1 (MCP-1) chemokine also known as CCL2. Monocyte chemoattractant protein and other inflammatory signaling molecules are thought to signal peripheral monocytes, neutrophils and lymphocytes to adhere to the NVU endothelial cell and eventually undergo chemotaxis into the brain and migrate to regions of injury ( Figure 14) [19].

Implications of White Blood Cell (Mononuclear) Lymphocyte Adherence in DBC Models
Adherence of the mononuclear white blood cell (WBC) (lymphocyte) in some NUVs of the diabetic DBC models have allowed not only the concept of an activated endothelium in DBC to be strongly considered as compared to CKC but also have stimulated the question as to the possibility that the aMGC may also be playing a role in signaling the WBC to come in contact with the EC and adhere to an activated endothelium ( Figure 13).
Previously, we had to include images of WBC adherence in Part I because there is such widespread multicellular remodeling that it is difficult to just examine one aspect of cellular remodeling in one image without including other cellular remodeling changes that concurrently occur in DBC models [1]. Also, these glial remodeling changes are associated with hyperglycemia and it is important to understand how hyperglycemia may drive the chronic neuroinflammation and the associated compromise of BBB TJ/AJ, which promote an increase in permeability and leads to memory loss and impaired cognition in T2DM mouse models in the diabetic DBC [18]. Activated microglia are known not only to contain certain cell surface receptors but also are responsible for the secretion of a specialized group of signaling cytokines and chemokines, which include monocyte chemoattractant protein 1 (MCP-1) chemokine also known as CCL2. Monocyte chemoattractant protein and other inflammatory signaling molecules are thought to signal peripheral monocytes, neutrophils and lymphocytes to adhere to the NVU endothelial cell and eventually undergo chemotaxis into the brain and migrate to regions of injury ( Figure 14) [19]. . Panel B demonstrates a surveilling ramified microglia (rMGC) and note how it is only surveilling the NVU for danger or damage signals. Also note the intact golden halo of astrocyte end feet surrounding the NVU. This is in contrast to the reactive-activated (aMGC), which appears to be invading the NVU. Note that in higher magnified images in panels (E-G) that the ECs also have aberrant mitochondria (depicted as yellow cores with encircling red lines). Also, note in (C,D) that there are some remaining intact astrocytes (iAC) to the right of the NVU (pseudo-colored golden); however to the left, note the absence of iACs adjacent to the outer basement membrane (BM) of the EC and pericyte of the NVU. Panel A depicts not only the adherence of a WBC to the activated EC but also depicts a region of microbleeds (encircled pseudo-colored yellow with yellow dashed line and the abnormal NVU with adherent WBC enclosed in a white line oval with pseudo-colored red interior. Panels (C-G) depict adhesion sites (asterisks) with a dashed line where the adherence appears to be continuous. The adherent mononuclear WBC cell is too small (2.5 × 4.5 μm) to be a monocyte and because of the thinned nearly absent cytoplasm is morphologically considered to represent a lymphocyte. Varying magnifications are present in the identifying boxes and the scale bars are present in the lower left of each image. 2;1;1;1 μm (panels (A-D) respectively) and 0.5 μm (panels (E,G) respectively).

Discussion
Previously, we have presented the data supporting concurrent multicellular maladaptive remodeling in the cells comprising the NVU structures I [1]. In the present paper we describe ultrastructural remodeling of activated microglia, which also contain aberrant mitochondria and NVU adherent WBCs to the activated luminal endothelial cell of the diabetic. Previously, we have posited that activated microglia may be related to accelerated aging and the increased risk of developing age-related neurodegeneration associated with T2DM (1). Furthermore, the ultrastructural changes noted in the DBC models may possibly contribute to the increased risk of developing age-related neurodegenerative disease and dementia such as sporadic Alzheimerʹs disease (AD) and Parkinsonʹs disease (PD) [1].
Mitochondrial dynamics are of critical importance in T2DM [20] and it has previously been documented that there is an increase in dynamin-1-like protein (Drp1) fission protein and increased activity of the glycogen synthase kinase 3 beta (GSK3β) proteins, while fusion proteins mitofusin-2 (Mfn2) and optic atrophy 1 (OPA-1) remain similar in T2DM as healthy controls [21,22]. Subsequently, the aberrant mitochondria are locked into a state of fission/fusion imbalance with Figure 14. Compilation of white blood cell adherence in diabetic DBC models. Panels (A,C-G) depict multiple increasing magnifications of a white blood cell (WBC) adhering to the luminal activated endothelial cell (EC). Panel B demonstrates a surveilling ramified microglia (rMGC) and note how it is only surveilling the NVU for danger or damage signals. Also note the intact golden halo of astrocyte end feet surrounding the NVU. This is in contrast to the reactive-activated (aMGC), which appears to be invading the NVU. Note that in higher magnified images in panels (E-G) that the ECs also have aberrant mitochondria (depicted as yellow cores with encircling red lines). Also, note in (C,D) that there are some remaining intact astrocytes (iAC) to the right of the NVU (pseudo-colored golden); however to the left, note the absence of iACs adjacent to the outer basement membrane (BM) of the EC and pericyte of the NVU. Panel A depicts not only the adherence of a WBC to the activated EC but also depicts a region of microbleeds (encircled pseudo-colored yellow with yellow dashed line and the abnormal NVU with adherent WBC enclosed in a white line oval with pseudo-colored red interior. Panels (C-G) depict adhesion sites (asterisks) with a dashed line where the adherence appears to be continuous. The adherent mononuclear WBC cell is too small (2.5 × 4.5 µm) to be a monocyte and because of the thinned nearly absent cytoplasm is morphologically considered to represent a lymphocyte. Varying magnifications are present in the identifying boxes and the scale bars are present in the lower left of each image. 2;1;1;1 µm (panels (A-D) respectively) and 0.5 µm (panels (E,G) respectively).

Discussion
Previously, we have presented the data supporting concurrent multicellular maladaptive remodeling in the cells comprising the NVU structures I [1]. In the present paper we describe ultrastructural remodeling of activated microglia, which also contain aberrant mitochondria and NVU adherent WBCs to the activated luminal endothelial cell of the diabetic. Previously, we have posited that activated microglia may be related to accelerated aging and the increased risk of developing age-related neurodegeneration associated with T2DM (1). Furthermore, the ultrastructural changes noted in the DBC models may possibly contribute to the increased risk of developing age-related neurodegenerative disease and dementia such as sporadic Alzheimer's disease (AD) and Parkinson's disease (PD) [1].
Mitochondrial dynamics are of critical importance in T2DM [20] and it has previously been documented that there is an increase in dynamin-1-like protein (Drp1) fission protein and increased activity of the glycogen synthase kinase 3 beta (GSK3β) proteins, while fusion proteins mitofusin-2 (Mfn2) and optic atrophy 1 (OPA-1) remain similar in T2DM as healthy controls [21,22]. Subsequently, the aberrant mitochondria are locked into a state of fission/fusion imbalance with mitochondrial fission being predominate. Further, these aberrant mitochondria may result in damage not only to the inner membranes but also the outer mitochondrial membranes that may allow for herniation and loss of the mitochondrial matrix proteins and sometimes result in mitochondria swelling ( Figure 15) [23].
Neuroglia 2018, 1 13 of 16 mitochondrial fission being predominate. Further, these aberrant mitochondria may result in damage not only to the inner membranes but also the outer mitochondrial membranes that may allow for herniation and loss of the mitochondrial matrix proteins and sometimes result in mitochondria swelling ( Figure 15) [23]. Recently, there has been an interest in hyperglycemia-driven neuroinflammation, which may compromise the integrity of BBB with increased permeability that leads to memory loss and impaired cognition [18]. This, along with the increasing interest in the vascular contributions to cognitive impairment and dementia, wherein scientific experts convened to discuss the research gaps in our understanding of how vascular factors contribute to Alzheimer's disease and related dementia has been discussed. [24,25]. These contributions to cognitive impairment and dementia could be considered inclusive of our current findings of the NVU remodeling including the glia ultrastructural remodeling in the diabetic DBC models. We propose that this terminology would be even more inclusive and put forth the title of microvascular contributions to cognitive impairment and dementia ( Figure 16). The clinical importance of which, may be discussed over the coming years and could possibly remain a constant when discussing the microvascular and glia remodeling of the brain as an end-organ in T2DM and diabetic cognopathy. The process of neuroinflammation including the activated microglia remodeling abnormalities in the DBC are also now candidates to be included in this microvascular contribution to cognitive impairment and dementia scenario. Importantly, a recent publication regarding the virtual kaleidoscopic presentation of identifying core gene microglial Recently, there has been an interest in hyperglycemia-driven neuroinflammation, which may compromise the integrity of BBB with increased permeability that leads to memory loss and impaired cognition [18]. This, along with the increasing interest in the vascular contributions to cognitive impairment and dementia, wherein scientific experts convened to discuss the research gaps in our understanding of how vascular factors contribute to Alzheimer's disease and related dementia has been discussed. [24,25]. These contributions to cognitive impairment and dementia could be considered inclusive of our current findings of the NVU remodeling including the glia ultrastructural remodeling in the diabetic DBC models. We propose that this terminology would be even more inclusive and put forth the title of microvascular contributions to cognitive impairment and dementia ( Figure 16). The clinical importance of which, may be discussed over the coming years and could possibly remain a constant when discussing the microvascular and glia remodeling of the brain as an end-organ in T2DM and diabetic cognopathy. The process of neuroinflammation including the activated microglia remodeling abnormalities in the DBC are also now candidates to be included in this microvascular contribution to cognitive impairment and dementia scenario. Importantly, a recent publication regarding the virtual kaleidoscopic presentation of identifying core gene microglial phenotypes and disease-specific microglial signatures has been shared, which aid in our trek into the future along with the concept of metabolic shifts and reprogramming of microglia [12,26].
phenotypes and disease-specific microglial signatures has been shared, which aid in our trek into the future along with the concept of metabolic shifts and reprogramming of microglia [12,26].
Indeed, these are exciting times having identified microglial core ultrastructural phenotype signatures consisting of amoeboid phenotypes, invasive NVU phenotypes, aberrant mitochondria and chromatin condensation-compaction as described by utilizing the established technique of TEM and the newer techniques of FIB/SEM. These established and newer techniques have allowed us to further explore some of the hidden secrets as put forth in part I and now this part II series of the microglia and mitochondria. Figure 16. Microvascular contribution to cognitive impairment and dementia. This cartoon image with transmission electron microscopic image inserts (a-d) depict impaired glucose tolerance (IGT), obesity and insulin resistance (IR) in diet-induced obesity (DIO) as the disease process progresses to increased glucotoxicity to overt type 2 diabetes mellitus (T2DM) in humans and our DBC models and may be associated with increasing cognitive impairment and dementia. As previously discussed, this is associated with a plethora of morphological ultrastructural multicellular remodeling changes and abnormalities, which include activated-amoeboid microglia cells (aMGC) (insert c) that may be associated with increased synthesis and secretion of functional toxic cytokines and chemokines in the DBC models. These abnormalities may contribute to neurodegeneration in addition to aMGC nuclear chromatin condensation (insert d) and leaky aberrant Mt (aMt) (insert a). These aMGCs may be associated and contribute to neuroinflammation, which may lead to the development of microvascular contributions to cognitive impairment and dementia (MVCID) and furthermore, may contribute to the development of sporadic Alzheimer's disease (SAD) and sporadic Parkinson's disease (PD). AGE = advanced glycation end products; BBB: Blood brain barrier; DIO: Diet-induced obesity; Inos: Inducible nitric oxide synthase; NOX = NADPH Ox (reduced nicotinamide adenine dinucleotide phosphate); NV: Neurovascular; RAGE: Receptor for advanced glycation endproducts; ROS: Reactive oxygen species; TJ/AJ: Tight junctions/adherens junctions; 3NT:3 nitrotyrosine; 4HNE: 4 hydroxynonenal; 8oxodG: 8-Oxo-2′-deoxyguanosine, which are biomarkers of oxidative stress for proteins, lipids, nucleic acids respectively.
On October 2nd, 2018, we found the following paper updating the role of microglia and feel that our readers should supplement our ultrastructural remodeling changes with a summary of the functional microglia changes put forth by Hammond T.R., Robinton D. and Stevens B. in their published preprint [27]. In the past few years there has been an explosion of functional information regarding microglia receptors and secreted proteins for surveillance and immune responses to better understand their multiple roles. It is now very clear amongst microglia researchers that microglia may assume a myriad of activation states or spectrum changes of activation ( Figure 2) from their development to their roles in forming ramified microglia to their spectrums of activated states, which Figure 16. Microvascular contribution to cognitive impairment and dementia. This cartoon image with transmission electron microscopic image inserts (a-d) depict impaired glucose tolerance (IGT), obesity and insulin resistance (IR) in diet-induced obesity (DIO) as the disease process progresses to increased glucotoxicity to overt type 2 diabetes mellitus (T2DM) in humans and our DBC models and may be associated with increasing cognitive impairment and dementia. As previously discussed, this is associated with a plethora of morphological ultrastructural multicellular remodeling changes and abnormalities, which include activated-amoeboid microglia cells (aMGC) (insert c) that may be associated with increased synthesis and secretion of functional toxic cytokines and chemokines in the DBC models. These abnormalities may contribute to neurodegeneration in addition to aMGC nuclear chromatin condensation (insert d) and leaky aberrant Mt (aMt) (insert a). These aMGCs may be associated and contribute to neuroinflammation, which may lead to the development of microvascular contributions to cognitive impairment and dementia (MVCID) and furthermore, may contribute to the development of sporadic Alzheimer's disease (SAD) and sporadic Parkinson's disease (PD). AGE = advanced glycation end products; BBB: Blood brain barrier; DIO: Diet-induced obesity; Inos: Inducible nitric oxide synthase; NOX = NADPH Ox (reduced nicotinamide adenine dinucleotide phosphate); NV: Neurovascular; RAGE: Receptor for advanced glycation endproducts; ROS: Reactive oxygen species; TJ/AJ: Tight junctions/adherens junctions; 3NT: 3 nitrotyrosine; 4HNE: 4 hydroxynonenal; 8oxodG: 8-Oxo-2 -deoxyguanosine, which are biomarkers of oxidative stress for proteins, lipids, nucleic acids respectively. Indeed, these are exciting times having identified microglial core ultrastructural phenotype signatures consisting of amoeboid phenotypes, invasive NVU phenotypes, aberrant mitochondria and chromatin condensation-compaction as described by utilizing the established technique of TEM and the newer techniques of FIB/SEM. These established and newer techniques have allowed us to further explore some of the hidden secrets as put forth in part I and now this part II series of the microglia and mitochondria.
On October 2nd, 2018, we found the following paper updating the role of microglia and feel that our readers should supplement our ultrastructural remodeling changes with a summary of the functional microglia changes put forth by Hammond T.R., Robinton D. and Stevens B. in their published preprint [27]. In the past few years there has been an explosion of functional information regarding microglia receptors and secreted proteins for surveillance and immune responses to better understand their multiple roles. It is now very clear amongst microglia researchers that microglia may assume a myriad of activation states or spectrum changes of activation ( Figure 2) from their development to their roles in forming ramified microglia to their spectrums of activated states, which includes synaptic pruning in health and disease and their supportive role of synaptic maintenance [6,27], Hammond T.R. et al. [27] state the following: "First, we must better understand the states that microglia assume in both development and disease. It is widely agreed that the term activation, or the bimodal M1/M2 scheme, does not sufficiently describe the multitude of ways in which these cells can respond to changes in their environment or the diversity of their functional states." Because of these evolving current concepts, we only discussed our observational ultrastructural remodeling changes of the microglia in relation to the neurovascular unit and other glial cells found in the female diabetic db/db models (DBC) and did not go into any depth regarding their functional changes as we were only performing ultrastructural remodeling changes. Indeed, these are exciting times to study the brain and microglial cells and reflect on the secret (the tissues [1]) to see how they undergo ultrastructural remodeling changes.