Oligodendrocyte and Myelin

neuroglia

Article Ultrastructural Remodeling of the Neurovascular Unit in the Female Diabetic db/db Model—Part III: Oligodendrocyte and Myelin

Melvin R. Hayden 1,2,*, Deana G. Grant 3, Aranyra Aroor 1,2,4 and Vincent G. DeMarco 1,2,4,5

1 Diabetes and Cardiovascular Center, University of Missouri School of Medicine, Columbia, MO 65212, USA; [email protected] (A.A.); [email protected] (V.G.D.) 2 Division of Endocrinology and Metabolism, Department of Medicine, University of Missouri, Columbia, MO 63212, USA 3 Electron Microscopy Core Facility, University of Missouri, Columbia, MO 65211, USA; [email protected] 4 Research Service, Harry S. Truman Memorial Veterans Hospital, Columbia, MO 65212, USA 5 Department of Medical Pharmacology and Physiology, University of Missouri, Columbia, MO 65212, USA * Correspondence: [email protected]; Tel.: +1-573-346-3019

Received: 9 October 2018; Accepted: 5 November 2018; Published: 8 November 2018

Abstract: Obesity, insulin resistance, and type 2 diabetes mellitus are associated with diabetic cognopathy. In this study, we tested the hypothesis that neurovascular unit(s) (NVU), oligodendrocytes, and myelin within cerebral cortical grey matter and deeper transitional zone regions between the cortical grey matter and white matter may be abnormal. The monogenic (Leprdb) female diabetic db/db [BKS.CgDock7m +/+ Leprdb/J] (DBC) mouse model was utilized for this ultrastructural study. Upon sacriﬁce (20 weeks of age), left-brain hemispheres of the DBC and age-matched non-diabetic wild type control C57BL/KsJ (CKC) mice were immediately immersion-ﬁxed. We found prominent remodeling of oligodendrocytes with increased nuclear chromatin condensation and volume and increased numbers of active myelination sites of the cytoplasm in transition zones. Marked dysmyelination with outer myelin lamellae sheath splitting, separation, and ballooning with aberrant mitochondria in grey matter and similar myelin remodeling changes with marked disarray with additional axonal collapse in transitional zones in DBC as compared to CKC models.

Keywords: db/db mouse model; myelin; neurovascular unit; oligodendrocyte; subcortical white matter; type 2 diabetes

1. Introduction Myelin is a multilayered lamellar sheath that enwraps axons in the grey and white matter in the brain and is synthesized in the brain by the oligodendroglia. The myelin sheath provides axonal protection and allows the saltatory conduction of the action potential. Oligodendrocytes (OLs), oligodendrocyte precursor cells (OPC), and oligodendrocyte lineage cells are specialized glial cells responsible for the synthesis, wrapping ensheathment, and compacting of myelin in myelinated axons [1–3]. Rudolf Ludwig Virchow (1821–1902) introduced the term myelin and defined it as medullary matter (Markstoff), or myeline, that in extremely large quantity fills up the interval between the axon axis-cylinder and the sheath in primitive nerve-fibers [4]. Michalski and Kothary [1] have set forth a paradigm for development of mature OLs in four distinct phases: (i) the birth, migration and proliferation of OPCs, that occurs in waves; followed by (ii) morphological differentiation with the OL establishing an expansive synthetic

Neuroglia 2018, 1, 351–364; doi:10.3390/neuroglia1020024 www.mdpi.com/journal/neuroglia Neuroglia 2018, 1 352 network of OL cytoplasmic processes; followed by (iii) axonal contact, which leads to myelin wrapping—ensheathment of targeted neuronal axons with ensuing generation of extremely electron Neuroglia 2018, 1, x FOR PEER REVIEW 2 of 14 dense and compact myelin with an enduring (iv) long-term trophic and metabolic support system for maintenancecytoplasmic and protection processes; of followed myelinated by (iii) neurons axonal [contact,1]. which leads to myelin wrapping— As OPCsensheathment differentiate of targeted into neuronal mature axons OLs with they ensuing undergo generation an of extensiveextremely electron development dense and of active compact myelin with an enduring (iv) long-term trophic and metabolic support system for cytoplasmicmaintenance reorganization and protection of cytoskeleton of myelinated neurons proteins [1]. and endoplasmic reticulum, which increases their intermediateAs OPCs cytoplasmic differentiate electroninto mature density OLs they and undergo helps an to extensive identify development them when of active studying with transmissioncytoplasmic electron reorganization microscopy of (TEM) cytoskeleton (Figure protei1). Identifyingns and endoplasmic characteristics reticulum, havewhich beenincreases presented in Part I Tabletheir 1 [5intermediate] as follows: cytoplasmic Oligodendrocytes electron density are an intermediated helps to identify in size them and when their studying thinned with cytoplasm transmission electron microscopy (TEM) (Figure 1). Identifying characteristics have been presented also have anin Part intermediate I Table 1 [5] as electron follows: Oligodendrocytes density that is helpfulare intermediate when in comparing size and their to thinned astrocytes cytoplasm and microglia. They alsoalso may have occur an inintermediate groups or electron nests anddensity are that more is helpful often foundwhen comparing in the white to astrocytes matter regionsand of the brain. In additionmicroglia. They to their also smallmay occur cytoplasmic in groups or volume, nests and theyare more have often multiple found in protoplasmic the white matter extensions, regions of the brain. In addition to their small cytoplasmic volume, they have multiple protoplasmic which allow OLs to concurrently myelinate multiple axons [1–4]. extensions, which allow OLs to concurrently myelinate multiple axons [1–4].

Figure 1. Comparison of astrocyte, ramified microglial cell, and oligodendrocyte to illustrate Figure 1. intermediateComparison electron of dens astrocyte,e and thin ramified cytoplasm. microglial(A) Depicts an cell, astrocyte and (AC) oligodendrocyte with electron lucent to illustrate intermediatecytoplasm electron and densea ramified and microglial thin cytoplasm. cell (rMGC) (withA) Depictsits electron an dense astrocyte cytoplasm. (AC) (B) with Illustrates electron a lucent cytoplasmthinned and a intermediate ramified microglial electron dense cell cytoplasm (rMGC) withof the its oligodendrocyte electron dense (OL) cytoplasm. obtained from (B )the Illustrates a thinned intermediatetransition zone electron in subcortical dense white cytoplasm matter (TZ of SCWM the oligodendrocyte) just deep to layer (OL)VI as compared obtained to from ACs and the transition microglia cells. Note that the OL is in the process of enwrapping at least 5 axons (1–5) with myelin in zone in subcortical(B) and its pseudo-colored white matter image, (TZSCWM) far right. Between just deep (A,B to) the layer cytoplasm VI as comparedhas been cut from to ACs each and cell microglia cells. Noteto that further the demonstrate OL is in the the processintermediate ofenwrapping electron (e) density at least of the 5 OL axons cytoplasm. (1–5) Also with note myelin the small in (B) and its pseudo-coloredamount image,of cytoplasm far right. as compared Between to nucleus (A,B) volume the cytoplasm as well as hasthe incorporation been cut from of myelin each at cell its to further demonstrateouter the margins intermediate of the cytoplasm electron plasmalemma. (e) density A ofpseudo-colorized the OL cytoplasm. image is Also inserted note far theright small to aid amount of in identifying OL nucleus (purple); cytoplasm (C green); fibrous astrocyte (fib AC blue); axons cytoplasm as compared to nucleus volume as well as the incorporation of myelin at its outer margins (yellow). Magnification ×3000; scale bar = 1 µm in A and 0.5 µm in (B). C: cytoplasm; MGC: microglial of the cytoplasmcell; M: myelin; plasmalemma. CKC: age-matched A pseudo-colorized non-diabetic wild image type control is inserted C57BL/KsJ. far right to aid in identifying OL nucleus (purple); cytoplasm (C green); fibrous astrocyte (fib AC blue); axons (yellow). Magnification ×3000; scaleThe bar small = 1cytoplasmicµm in A and volume 0.5 µ ofm oligodendrocyte in (B). C: cytoplasm; may be MGC: related microglial to its function cell; of M: constantly myelin; CKC: supplying its plasma membrane (up to three-times its volume of plasma membranes) in order to age-matched non-diabetic wild type control C57BL/KsJ. create the multiple myelin lamellar sheaths. These myelin lamella that enwrap multiple unmyelinated axons increase the speed of neuronal transmission of their action potentials via The smallmembrane cytoplasmic depolarization volume by high-density of oligodendrocyte voltage-gated sodium may be channels related that to creates its function the saltatory of constantly supplying(jumping) its plasma conduction membrane of their (up action to three-times potentials and its signaling volume at of the plasma nodes membranes)of Ranvier (Figure in order 1B). to create Importantly, this saltatory conduction allows electrical nerve signals to be propagated long distances the multipleat high myelin rates lamellarwithout any sheaths. degradation These of the myelin action lamellapotential thatsignaling enwrap [1–4]. multiple unmyelinated axons increase the speedMyelin of also neuronal serves as transmissiona protective sheath of theirof myelinated action potentialsneurons in order via membraneto provide for depolarizationlong- by high-densityterm axonal voltage-gated integrity and sodiummaintenance channels survival. thatMyelinated creates axons the are saltatory present in (jumping) the cortical conductiongrey of their actionmatter; potentials however, and they signalingare the most atabundant the nodes within of the Ranvier white matter (Figure and give1B). rise Importantly, to the white matter this saltatory tracts. The bulk of myelinated axons localizes within the white matter, which are important for conductioncarrying allows large electrical amounts nerve of information signals tofrom be one propagated region of the long brain distances to distant atregions high and rates must without any degradationrapidly of the transmit action this potential information. signaling This rapid [1 transmission–4]. is primarily due to its compacted electron Myelin also serves as a protective sheath of myelinated neurons in order to provide for long-term axonal integrity and maintenance survival. Myelinated axons are present in the cortical grey matter; however, they are the most abundant within the white matter and give rise to the white matter tracts. The bulk of myelinated axons localizes within the white matter, which are important for carrying large amounts of information from one region of the brain to distant regions and must rapidly transmit this Neuroglia 2018, 1 353 information. This rapid transmission is primarily due to its compacted electron dense myelin sheaths. Therefore, if myelin undergoes any significant abnormal remodeling change there may be a delay in the in the arrival of information to the more distant regional neurons with subsequent cognitive impairment [6,7].

2. MaterialsNeuroglia and 2018 Methods, 1, x FOR PEER REVIEW 3 of 14 dense myelin sheaths. Therefore, if myelin undergoes any significant abnormal remodeling change 2.1. Animalthere Models may be a delay in the in the arrival of information to the more distant regional neurons with subsequent cognitive impairment [6,7]. As previously presented in Part I [5]: All animal studies were approved by the Institutional Animal Care2. Materials and Use and Committees Methods at the Harry S Truman Memorial Veterans’ Hospital and University of Missouri, Columbia, MO, USA (No. 190), and conformed to the Guide for the Care and Use of 2.1. Animal Models Laboratory Animals published by the National Institutes of Health (NIH). Eight-week-old female db/db [BKS.Cg-As previouslyDock7m +/+ presentedLepr dbin/ J]Part (DBC) I [5]: All and animal wild-type studies were control approved C57BLKS/J by the Institutional (CKC) mice were Animal Care and Use Committees at the Harry S Truman Memorial Veterans’ Hospital and purchasedUniversity from the of JacksonMissouri, Columbia, Laboratory MO, (AnnUSA (No. Harbor, 190), and MI, conformed USA) and to the were Guide housed for the Care under and standard ◦ laboratoryUse conditions of Laboratory where Animals room publishe temperatured by the was National 21–22 InstitutesC, and oflight Health and (NIH). dark Eight-week-old cycles were 12 h each. Two cohortsfemale of micedb/db were[BKS.Cg- used:Dock7 leanm +/+ non-diabeticLeprdb/J] (DBC) and controls wild-type (CKC, controln C57BLKS/J= 3), and (CKC) obese, mice insulin-resistant, were diabetic db/dbpurchased (DBC, fromn the= 3),Jackson which Laboratory were sacriﬁced (Ann Harb foror, MI, study USA) at and 20 weekswere housed of age. under standard laboratory conditions where room temperature was 21–22 °C, and light and dark cycles were 12 h each. Two cohorts of mice were used: lean non-diabetic controls (CKC, n = 3), and obese, insulin- 2.2. Tissue Collection and Preparation resistant, diabetic db/db (DBC, n = 3), which were sacrificed for study at 20 weeks of age. Have been previously presented in Part I [5]. 2.2. Tissue Collection and Preparation 3. Results Have been previously presented in Part I [5].

3.1. Dysmyelination3. Results in the Cortical Grey Matter Primarily Layer III Splitting3.1. Dysmyelination and separation in the ofCortical myelin Grey lamellar Matter Primarily sheaths Layer (dysmyelination) III in the cortical grey matter layer III was readilySplitting observedand separation in theof myelin female lamellar diabetic sheaths db/db (dysmyelination) [BKS.Cg Dock7in the corticalm +/+ greyLepr matterdb/J] (DBC) as m db comparedlayer to the III nondiabeticwas readily observed age-matched in the female non-diabetic diabetic db/db wild [BKS.Cg type controlDock7 +/+ C57BL/KsJ Lepr /J] (DBC) (CKC) as models. compared to the nondiabetic age-matched non-diabetic wild type control C57BL/KsJ (CKC) models. Additionally,Additionally, axonal remodelingaxonal remodeling was was noted, noted, which which consisted of aberrant of aberrant mitochondria mitochondria and increased and increased axonal cytoplasmaxonal cytoplasm electron electron density density as comparedas comparedto to the nondiabetic nondiabetic CKC CKC model model (Figures (Figures 2 and 3).2 and3).

m db Figure 2. ComparisonFigure 2. Comparison of control of control CKC CKC and and diabetic diabetic diabetic diabetic db/db db/db [BKS.Cg [BKS.CgDock7Dock7 +/+ Leprm +/+/J] (DBC)Leprdb /J] (DBC) myelination in layer III cortical grey matter. These panels depict the marked difference between the myelinationmyelination in layer of III axons cortical in the grey cortical matter. grey matter These of Layer panels III.depict Note in thethe diabetic marked DBC difference panel (to the between the myelinationright) of the axons marked in splitting the cortical and separation grey matter of mye oflin Layer(dysmyelination) III. Note and in the grouping diabetic of DBCsuspected panel (to the right) the markedaberrant mitochondria splitting and (encircled separation white ofdashed myelin line). (dysmyelination) The insert in the DBC and panel the is grouping from another of suspected aberrant mitochondriamodel in cortical (encircledlayer III with white dysmyelination. dashed line).Also note The the insert increased in thethickness DBC of panel myelin iswhich from another measures approximately 0.5 µm in the DBC as compared to the 0.1 µm thickness of the myelin in the model in corticalCKC model. layer Also III note with the elongated dysmyelination. axonal mitochondr Alsoia note in the the CKC increased model in contrast thickness to the of smaller myelin which measuresrounded approximately mitochondria 0.5 inµm the in DBC. the Importantly, DBC as compared note the increase to the in 0.1 electronµm thicknessdensity of the of axoplasm the myelin in the CKC model.of the Also neuron note in thethe DBC elongated as compared axonal to the mitochondria CKC. Magnification in the ×2000; CKC bar model = 1 µm. in Magnification contrast to of the smaller rounded mitochondriainsert ×4000 and inscale the bar DBC. = 0.5 µm. Importantly, note the increase in electron density of the axoplasm of the neuron in the DBC as compared to the CKC. Magniﬁcation ×2000; bar = 1 µm. Magniﬁcation of insert ×4000 and scale bar = 0.5 µm. Neuroglia 2018, 1 354

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Figure 3. Dysmyelination and aberrant mitochondria in cortical grey matter layer III of diabetic DBC Figure 3.models.Dysmyelination (A) Depicts the and normal aberrant compact mitochondria myelin (M) ensh ineathing cortical the grey axons matter of the layernondiabetic III of control diabetic DBC models.CKC (A) models Depicts and the note normal the normal compact electron myelin dense (M)mito ensheathingchondria (Mt) (encircled the axons by of yellow the nondiabetic dashed line). control CKC modelsPanels and(B,C) notedepict the thenormal marked electronsplitting and dense separation mitochondria of myelin (Mt)ensheathment (encircled in byDBC yellow models dashed FigureFigure 3. Dysmyelination 3. Dysmyelination and and aberrant aberrant mitochondria mitochondria inin cocorticalrtical grey grey matter matter laye layer IIIr of III diabetic of diabetic DBC DBC (arrows).models. Also (A) Depicts note the the aberrant normal compact Mt (aMt)—(pse myelin (M)udo-colored ensheathing yellow the axons with of redthe nondiabeticlines) and controlincreased line).models. Panels (AB,)C Depicts) depict the the normal marked compact splitting myelin and (M) separation ensheathing of myelinthe axons ensheathment of the nondiabetic in DBC control models electronCKC models dense cytoplasmand note the in normal the DB electronC. Magnification dense mito ×4000;chondria bar (Mt) = 0.5 (encircled µm. by yellow dashed line). (arrows).CKC models Also note and note the aberrantthe normal Mt electron (aMt)—(pseudo-colored dense mitochondria (Mt) yellow (encircled with by red yellow lines) dashed and increasedline). Panels (B,C) depict the marked splitting and separation of myelin ensheathment in DBC models electron dense cytoplasm in the DBC. Magniﬁcation ×4000; bar = 0.5 µm. PanelsAlso,(arrows). (B ballooning,C) depict Also note theof themarkedmyelinated aberrant splitting Mt axons (aMt)—(pse and was separation udo-coloreddetecte d,of butyellowmyelin not with ensheathmentwith red thelines) frequency and in increased DBC ofmodels myelin splitting(arrows).electron and Also separation dense note cytoplasm the (Figure aberrant in the 4). Mt DB C.(aMt)—(pse Magnificationudo-colored ×4000; bar =yellow 0.5 µm. with red lines) and increased Also,electron ballooning dense ofcytoplasm myelinated in the DB axonsC. Magnification was detected, ×4000; but bar not = 0.5 with µm. the frequency of myelin splitting Also, ballooning of myelinated axons was detected, but not with the frequency of myelin and separation (Figure4). Also,splitting ballooning and separation of myelinated (Figure 4). axons was detected, but not with the frequency of myelin splitting and separation (Figure 4).

Figure 4. Examples of ballooning in myelinated axons of diabetic DBC models in cortical grey matter. This three-panel image depicts ballooning of myelinated axons that were only observed in the diabetic DBCFigure and 4.did Examples not occur of ballooningin nondiabetic in myelinated control CKC. axons The of diabetic left-hand DBC panel models depicts in cortical a myelinated grey matter. axon Figure 4. Examples of ballooning in myelinated axons of diabetic DBC models in cortical grey matter. withThis ballooning three-panel superiorly image depicts (open balloon arrowing 1) of and myelinated a ballooning axons inferiorlythat were only (open observed arrow in2) the and diabetic note the This three-panelinterruptionDBC and image didof the not depicts myelin occur in sheath ballooning nondiabetic in mid ofcontrol axon myelinated (redCKC. dashed The axonsleft-hand oval) that an paneld werethe depicts cropped only a observedmyelinated image insert inaxon from the diabetic DBCFigure andthis didwith region4. Examples notballooning exploded occur of superiorly in ballooning in nondiabetic Microsoft (open in myelinatedarrowpaint control with1) and CKC.in axons atact ba llooningscale Theof diabetic left-handbar inferiorly of DBC2 µm. panel(open models Mid arrow depictspanel in cortical 2) has and a open myelinatednote grey arrowthe matter. axon This three-panelinterruption imageof the myelindepicts sheath balloon in ingmid of axon myelinated (red dashed axons oval) that and were the cropped only observed image insert in the from diabetic with ballooningpointing to superiorly balloon 1 and (open right-hand arrow panel 1) and has aopen ballooning arrow pointing inferiorly to balloon (open 2. Note arrow the 2) aberrant and note the DBCmitochondria andthis regiondid not explodedpseudo-colored occur in in nondiabetic Microsoft yellow paint with control with red outline.CKC.intact scaleThe Magnification left-handbar of 2 µm. panel ×1500; Mid depicts panelscale barhas a myelinated =open 2 µm arrow far left, axon interruptionpointing of the to myelin balloon sheath1 and right-hand in mid axonpanel (redhas open dashed arrow oval) pointing and to the balloon cropped 2. Note image the aberrant insert from this withMagnification ballooning ×4000;superiorly scale (openbar = 0.5 arrow µm in 1) mid and and a ba farllooning right-hand inferiorly panels. (open arrow 2) and note the µ regioninterruption explodedmitochondria inof Microsoftthe pseudo-colored myelin paintsheath withyellow in mid intact with axon red scale (redoutline. bar dashed Magnification of 2 oval)m. Midan ×1500;d the panel croppedscale has bar open =image 2 µm arrow farinsert left, pointing from to Magnification ×4000; scale bar = 0.5 µm in mid and far right-hand panels. balloonthisAn 1 region and unexpected right-hand exploded finding in panel Microsoft within has open paintthe cortical arrow with pointingin grtactey matterscale to bar balloon consisted of 2 µm. 2. Noteof Mid fragmented thepanel aberrant has microtubules— open mitochondria arrow pseudo-colored yellow with red outline. Magnification ×1500; scale bar = 2 µm far left, Magnification neurofilamentspointingAn tounexpected balloon and electron 1 findingand right-hand dense within proteinaceous the panel cortical has gropen aeyggregations matter arrow consistedpointing in three toof differentballoonfragmented 2. unmyelinated Note microtubules— the aberrant axons ×4000;at mitochondriahigherneurofilaments scale magnifications bar = pseudo-colored 0.5 andµ melectronin (Figure mid dense yellow and 5). proteinaceous far with right-hand red outline. aggregations panels. Magnification in three ×1500; different scale unmyelinated bar = 2 µm far axons left, Magnificationat higher magnifications ×4000; scale (Figure bar = 0.5 5). µm in mid and far right-hand panels. An unexpected finding within the cortical grey matter consisted of fragmented An unexpected finding within the cortical grey matter consisted of fragmented microtubules— microtubules—neurofilaments and electron dense proteinaceous aggregations in three different neurofilaments and electron dense proteinaceous aggregations in three different unmyelinated axons unmyelinatedat higher magnifications axons at higher (Figure magnifications 5). (Figure5).

Figure 5. Larger axons, loss of microtubule neuroﬁlament linearity, fragmentation and proteinaceous

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aggregation and deposition within axoplasm in some of diabetic DBC models. (B,C) Depict a loss of linearity of neuroﬁlaments (nf) with fragmentation and aggregated proteinaceous electron dense deposits (within the lower half, below the red dashed line) axoplasm of diabetic DBC as compared to nondiabeticNeuroglia CKC 2018, 1 controls, x FOR PEER (A REVIEW). Some of the larger electron densities are microtubules (MT), while5 of 14 others

could be proteinFigure 5. aggregations. Larger axons, loss (B of,C microtubule) Are from neurofila differentment regions linearity, of fragmentation the same unmyelinatedand proteinaceous axon. Note that the axonaggregation width of and the deposition DBC is ~1.5withinµ maxoplasm as compared in some of to diabetic 0.5 µm DBC in CKC. models. Magnifications (B,C) Depict a loss×10,000 of (A,C); scale bar =linearity 0.2 µm. of (neurofilamentsB) Magnifications (nf) with×6000; fragmentatio scale barn and = 0.5aggregatedµm. proteinaceous electron dense deposits (within the lower half, below the red dashed line) axoplasm of diabetic DBC as compared to nondiabetic CKC controls (A). Some of the larger electron densities are microtubules (MT), while others could be protein aggregations. (B,C) Are from different regions of the same unmyelinated 3.2. Dysmyelinationaxon. Note in thethat Transitionalthe axon width of Zone the DBC between is ~1.5 µm Cortical as compared Layer to VI 0.5 andµm in White CKC. Magnifications Matter ×10,000 (A,C); scale bar = 0.2 µm. (B) Magnifications ×6000; scale bar = 0.5 µm. Due to the observations of splitting and separation of myelin lamellar units in the cortical grey matter, we3.2. decided Dysmyelination to make in the deeper Transitional cuts Zone to between see if Cortical the transitional Layer VI and White zone Matter (TZ) between cortical layer and subcorticalDue white to the matterobservations also of demonstrated splitting and separation abnormalities of myelin lamellar in myelinated units in the axons cortical since grey we were unable tomatter, examine we decided any specific to make white deeper mattercuts to see tracts. if the transitional One of the zone first (TZ) things between we cortical noted layer was and a decrease in neurovascularsubcortical units white inmatter these also regions demonstrated as compared abnormalities to in cortical myelinated grey axons matter. since we These were unable findings were anticipated,to examine since it any is known specific thatwhitethe matter white tracts. matter One of has the decreasedfirst things we capillary noted was density a decrease as compared in to neurovascular units in these regions as compared to cortical grey matter. These findings were cortical greyanticipated, matter since regions it is [known8]. that the white matter has decreased capillary density as compared to We notedcortical a grey marked matter difference regions [8]. in myelinated axons in the TZ in the DBC models. These regions were observedWe to noted have a amarked marked difference disarray in myelinated of myelin axons with in splitting—separationthe TZ in the DBC models. andThese collapse regions of axons from the myelinwere observed lamellar to have sheaths a marked (Figure disarray6). of myelin with splitting—separation and collapse of axons from the myelin lamellar sheaths (Figure 6).

Figure 6. Extensive myelin remodeling disarray in diabetic DBC at deeper transition regions of the Figure 6. Extensivesubcortical white myelin matter remodeling regions. (A) Illustrates disarray the in normal diabetic appearance DBC at ofdeeper the transitional transition subcortical regions of the subcorticalwhite white matter matter regions regions. just deep (A )to Illustrates layer VI grey the matter. normal (B,C appearance) Depict markedly of the abnormal transitional myelin subcortical white matterremodeling regions in the just diabetic deep DBC to layermodels. VI (B) grey Even matter.demonstrates (B, Ca demarcation) Depict markedly between more abnormal severe myelin remodeling to the left side of the demarcation (yellow dashed line) with more severe myelin splitting remodeling in the diabetic DBC models. (B) Even demonstrates a demarcation between more severe and axonal—axoplasm collapse (open red arrows) within the abnormal myelin lamellar sheaths. (C) remodelingDepicts to the a centrally left side located of the oligodendrocyte demarcation (OL (yellow outlined dashed by yellow line) dashed with line) more that severeappears to myelin be in splitting and axonal—axoplasmthe process of wrapping collapse multiple (open axons red with arrows) myelin. within Importantly, the abnormal note the very myelin electron lamellar dense sheaths. (C) Depictschromatin a centrally clumping—condensation located oligodendrocyte within the nuclei (OL and outlined the very bysmall yellow amount dashed of cytoplasm. line) (B that,C) appears to be in theDepict process marked of disorganization wrapping multiple of myelin axonslamella—dysmyelination with myelin. with Importantly, disordered notemyelin the lamella, very electron which is associated with splitting and separation of the myelin sheath lamella. Also, this region dense chromatindemonstrated clumping—condensation marked abnormal remodeling within chan theges nuclei of myelinated and the neurons very smallin the amountdiabetic DBC of cytoplasm. (B,C) Depictmodels marked as compared disorganization to control CKC ofmode myelinls. Magnification lamella—dysmyelination ×2000; scale bar = 1 µm ( withA). Magnification disordered myelin lamella, which×1200; is scale associated bar = 1 µm with (B,C splitting). and separation of the myelin sheath lamella. Also, this region demonstrated marked abnormal remodeling changes of myelinated neurons in the diabetic DBC Also, there were abnormalities of OLs in the diabetic DBC models in the transitional zone × µ modelssubcortical as compared white to matter control between CKC layers models. VI Magniﬁcationand deeper white 2000;matter scale (Figures bar 7 = and 1 m8). (A). Magniﬁcation ×1200; scale bar = 1 µm (B,C).

Also, there were abnormalities of OLs in the diabetic DBC models in the transitional zone subcortical white matter between layers VI and deeper white matter (Figures7 and8). Neuroglia 2018, 1 356 Neuroglia 2018, 1, x FOR PEER REVIEW 6 of 14

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Figure 7. Increased chromatin condensation volume in nuclei of diabetic DBC models in the Figure 7. Increased chromatin condensation volume in nuclei of diabetic DBC models in the transitional transitional zone subcortical regions as compared to nondiabetic CKC models. The nondiabetic CKC zone subcortical regions as compared to nondiabetic CKC models. The nondiabetic CKC model to modelFigure to7. the Increased left (cytoplasm chromatin pseudo-colored condensation green volume and outlinedin nuclei with of diabeticwhite dashed DBC line)models depicts in thean the left (cytoplasm pseudo-colored green and outlined with white dashed line) depicts an OL in OLtransitional in the transitional zone subcortical zone regionssubcortical as compared (TZSC) regionto nondiabetic with surrounding CKC mode ls.myelinated The nondiabetic axons CKCand the transitionaldisplaysmodel to atthe zoneleast left (cytoplasm subcortical1–7 numbered pseudo-colored (TZSC) regions region of greenmye withlination and surrounding outlined and maintenance with myelinated white dashed zones. axons line)Note and depicts that displays the an at leastcytoplasmOL 1–7 in numbered the (C)transitional in regions control zone ofmodels myelinationsubcortical to the (TZSC)left and has maintenance regionapproximately with zones.surrounding seven Note regions myelinated that of the myelination cytoplasm axons and as (C) in controlcompareddisplays models at to toleast diabetic the 1–7 left DBCnumbered has model approximately regionsto the right, of sevenmye whichlination regions depicts and of up maintenance myelination to 16 regions zones. as of compared myelination Note that to and diabeticthe DBCmaintenancecytoplasm model to the(C) zones right,in control with which adjacent models depicts numerous to upthe toleft electron 16 has regions approximately dense of myelinationmyelinated seven axons regions and (cytoplasm maintenance of myelination is pseudo- zones as with adjacentcoloredcompared numerous red to with diabetic electronwhite DBC dashed dense model outline). myelinated to the Of right, great axonswhich interest (cytoplasmdepicts in the up TZSC to is 16 regions pseudo-colored regions of ofthe myelination DBC, red note with thatand white dashednuclearmaintenance outline). chromatin Ofzones great (Chr) with interest is adjacent markedly in numerous the increased TZSC electron regions (outlined dense of by the yellow myelinated DBC, dashed note axons that lines) nuclear (cytoplasm in volume chromatin iscompared pseudo- (Chr) is to nuclear volume when compared to the CKC nuclear chromatin on the left (outlined by yellow markedlycolored increased red with (outlinedwhite dashed by yellowoutline). dashed Of great lines) interest in volumein the TZSC compared regions toof the nuclear DBC, volumenote that when dashed lines). This was a consistent remodeling abnormality in the TZSC regions in the DBC models. comparednuclear to chromatin the CKC (Chr) nuclear is markedly chromatin increased on the (out leftlined (outlined by yellow by dashed yellow lines) dashed in volume lines). compared This was a Magnificationto nuclear volume ×2000; when bar = compared 0.5 µm in CKCto the and CKC 1 µm nu clearin DBC. chromatin on the left (outlined by yellow consistent remodeling abnormality in the TZSC regions in the DBC models. Magniﬁcation ×2000; dashed lines). This was a consistent remodeling abnormality in the TZSC regions in the DBC models. bar = 0.5 µm in CKC and 1 µm in DBC. Magnification ×2000; bar = 0.5 µm in CKC and 1 µm in DBC.

Figure 8. Markedly abnormal subcortical white matter myelin in diabetic DBC models. (C,D) Depict marked disorganization—disarrangement of the transitional zone subcortical white matter (SCWM) Figure 8. Markedly abnormal subcortical white matter myelin in diabetic DBC models. (C,D) Depict

Figuremarked 8. Markedly disorganization—disarrangement abnormal subcortical white of the matter transitional myelin zone in subcortical diabetic DBC white models. matter (SCWM) (C,D) Depict marked disorganization—disarrangement of the transitional zone subcortical white matter (SCWM)

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Neuroglia 2018, 1, x FOR PEER REVIEW 7 of 14 myelin and the neuronal axon in the diabetic DBC models. (B) Illustrates axon-axoplasm collapse (doubleNeuroglia 2018 arrows), 1, x FOR in myelinated PEER REVIEW neurons. Also note that the neuronal axons become very electron7 dense of 14 myelin and the neuronal axon in the diabetic DBC models. (B) Illustrates axon-axoplasm collapse once they collapse (pseudo-colored yellow and outlined by yellow dashed lines). Additionally, note the (doublemyelin arrows) and thein myelinated neuronal axon neurons. in the Alsodiabetic note DBC that models. the neuronal (B) Illustrates axons becomeaxon-axoplasm very electron collapse dense aberrant mitochondria (pseudo-colored yellow and outlined by red lines) as compared to the electron once (doublethey collapse arrows) (pseudo-colored in myelinated neurons. yellow Also and note outlined that the neuronalby yellow axons dashed become lines). very Additionally, electron dense note dense Mt with crista in the control CKC models (A,C). (D) Illustrates the abnormal myelin lamella the aberrantonce they mitochondria collapse (pseudo-colored (pseudo-colored yellow andyellow outlined and by outlined yellow dashedby red lines). lines) Additionally, as compared note to the with marked splitting and separation of myelin lamella. Magniﬁcation ×4000; bar = 0.5 µm in (A,B). electronthe aberrantdense Mt mitochondria with crista (pseudo-coloredin the control CKC yellow models and outlined (A,C). (byD) redIllustrates lines) as the compared abnormal to the myelin Magniﬁcation ×12,000; bar = 200 nm in (C,D). art: artifact. lamellaelectron with densemarked Mt splitting with crista and in theseparation control CKC of myelin models lamella. (A,C). ( DMagnification) Illustrates the ×4000; abnormal bar myelin= 0.5 µm in (A,B).lamella Magnification with marked ×12,000; splitting bar and = 200 separation nm in ( Cof, Dmyelin). art: lamella. artifact. Magnification ×4000; bar = 0.5 µm in (A,B). Magnification ×12,000; bar = 200 nm in (C,D). art: artifact. Additionally noted noted was was the the collapse of of the axon axonss with myelin splitting and separation in the Additionally noted was the collapse of the axons with myelin splitting and separation in the diabeticdiabetic DBC DBC models models (Figures (Figures 99 and and9 and 10 10). 10).).

Figure 9. Axonal collapse in dysmyelinated neurons with splitting and separation transitional zones Figure 9. Axonal collapse in dysmyelinated neurons with splitting and separation transitional zones in Figurein 9.diabetic Axonal DBC. collapse Note inin dysmyelinated(B) that the collapse neurons of the with axon splitting within the and dysmyelinated separation transitional neuron with zones diabetic DBC. Note in (B) that the collapse of the axon within the dysmyelinated neuron with splitting in diabeticsplitting DBC. and separation Note in (ofB )myelin that the lamella collapse sheath of in the transitionalaxon with zonesin the (TZ) dysmyelinated of the subcortical neuron white with and separation of myelin lamella sheath in the transitional zones (TZ) of the subcortical white matter splittingmatter and of separation the diabetic of DBC myelin as compared lamella sheath to the TZ in thenon-collapsed transitional axons zones in (TZ)CKC ofmodels the subcortical (A). Axonal white of the diabetic DBC as compared to the TZ non-collapsed axons in CKC models (A). Axonal collapse mattercollapse of the was diabetic markedly DBC increased as compared in the transitionalto the TZ non-collapsedzone regions as comparedaxons in CKCto the modelsgrey matter (A). and Axonal was markedly increased in the transitional zone regions as compared to the grey matter and not found collapsenot wasfound markedly in any of increasedthe CKC models. in the transitionalNote the region zone of regionsthe TZ in as artistic compared rendering to the to grey far right matter of and in anyabove of the figure. CKC Magnification models. Note ×5000; the scale region bar of = the0.5 µm. TZ in artistic rendering to far right of above ﬁgure. not found in any of the CKC models. Note the region of the TZ in artistic rendering to far right of Magniﬁcation ×5000; scale bar = 0.5 µm. above figure. Magnification ×5000; scale bar = 0.5 µm.

Figure 10. Myelin splitting, separation, and ballooning with axonal collapse in transitional zone in diabetic DBC models. (A) Depicts myelinated neurons with pseudo-colored green axons that fully occupy the myelin core. Also note the pseudo-colored yellow fibrous astrocyte (f AC) that are adjacent Figureto 10.the Myelinmyelinated splitting, axons. separation,(B) Demonstrates and ballooningthe splitting, wi separath axonaltion, andcollapse ballooning in transitional of the myelin zone in Figure 10. Myelin splitting, separation, and ballooning with axonal collapse in transitional zone in diabeticsheath DBC resulting models. in axonal(A) Depicts collapse myelinated in the diabetic neurons DBC models with inpseu the do-coloredtransitional zonegreen between axons layers that fully diabetic DBC models. (A) Depicts myelinated neurons with pseudo-colored green axons that fully occupyVI theand myelinthe subcortical core. Also white note matter. the pseudo-colored Magnification ×3000; yellow scale fibrous bar = 1 astrocyteµm in (A,B (f). AC) that are adjacent occupy the myelin core. Also note the pseudo-colored yellow ﬁbrous astrocyte (f AC) that are adjacent to the myelinated axons. (B) Demonstrates the splitting, separation, and ballooning of the myelin to4. Discussion the myelinated axons. (B) Demonstrates the splitting, separation, and ballooning of the myelin sheath resulting in axonal collapse in the diabetic DBC models in the transitional zone between layers sheath resulting in axonal collapse in the diabetic DBC models in the transitional zone between layers VI andWhite the subcortical matter is usually white matter. thought Ma tognification be composed ×3000; primarily scale bar by = 1myelinated µm in (A,B ).axons at the gross VI and the subcortical white matter. Magniﬁcation ×3000; scale bar = 1 µm in (A,B). macroscopic level and is thought to be responsible for imparting a demarcation from the outer grey 4. Discussionmatter, which contains primarily neuronal cell bodies, glia, synaptic dendrites, and axon terminals 4. Discussionof neurons. However, the white matter also contains numerous and varying percentages of White matter is usually thought to be composed primarily by myelinated axons at the gross Whiteunmyelinated matter axons: is usually for example, thought the to human be composed corpus callosum primarily may by have myelinated up to 30% axonsunmyelinated at the gross macroscopic level and is thought to be responsible for imparting a demarcation from the outer grey macroscopicaxons in contrast level and to the is thought optic nerve to bewhere responsible nearly all foraxons imparting are myelinated a demarcation [9]. from the outer grey matter, which contains primarily neuronal cell bodies, glia, synaptic dendrites, and axon terminals matter, which contains primarily neuronal cell bodies, glia, synaptic dendrites, and axon terminals of of neurons. However, the white matter also contains numerous and varying percentages of neurons. However, the white matter also contains numerous and varying percentages of unmyelinated unmyelinated axons: for example, the human corpus callosum may have up to 30% unmyelinated axons in contrast to the optic nerve where nearly all axons are myelinated [9].

Neuroglia 2018, 1 358 axons: for example, the human corpus callosum may have up to 30% unmyelinated axons in contrast Neuroglia 2018, 1, x FOR PEER REVIEW 8 of 14 to the optic nerve where nearly all axons are myelinated [9]. Myelin wrapping and the final ﬁnal end-product end-product of hi highlyghly electron electron dense and and compacted compacted myelination myelination of axonal ensheathment is quite complicated and and illustrated illustrated in in a a simplified simpliﬁed illustration illustration (Figure (Figure 11).11). One can also view a more detailed 3D reconstructionreconstruction with electron microscopy in the following reference for greater detail [[10]10].

Figure 11. Possible mechanisms for oligodendrocyte myelination. (A) Depicts an illustration of the Figure 11. Possible mechanisms for oligodendrocyte myelination. (A) Depicts an illustration of the initial engagement of an OL and an unmyelinated axon with a protrusion of an oligodendrocyte initial engagement of an OL and an unmyelinated axon with a protrusion of an oligodendrocyte cytoplasmic process (grey) beginning to extend and enwrap (blue dashed lines) of an unmyelinated cytoplasmic process (grey) beginning to extend and enwrap (blue dashed lines) of an unmyelinated neuron. ( (BB)) Depicts Depicts the the possible possible multi-step process: (1) (1) solid solid grey oligodendrocyte cytoplasmic process protrusion and engagement of the OL to th thee unmyelinated axon; (2) extension of the original oligodendrocyte cytoplasmic protru protrusion—bluesion—blue dashed dashed line; line; (3) (3) anot anotherher wrapping—red dashed dashed line; line; (4) the most recent extension of wrapping—green dashed line. These previous steps may represent a common OLOL cellular cellular mechanism mechanism of myelination. of myelination. The multiple The multiple wrappings, wrappings, which result which in multilamellar result in multilamellarmyelin ensheathments myelin ensheathments are then bound are tightly then and bound compacted tightly viaand the co myelinmpacted basic via proteinthe myelin plus otherbasic proteincompacting plus proteinsother compacting synthesized prot andeins secreted synthesized by oligodendrocytes and secreted by oligodendrocytes that eventually develop that eventually into the develophighly electron into the dense highly tightly electron wrapped dense compacted tightly wrapped myelinated compacted neurons that myelinated we observe neurons in transmission that we observeelectron in microscopic transmission images electron in microscopic control CKC images models in ascontrol depicted CKC in models the control as depicted CKC modelin the control in (C). CKCThis mechanismmodel in (C). does This not mechanism explain for does the not lateral explain extension for the oflateral the internodalextension of myelin. the internodal Note that myelin. in (C) Notethere isthat present in (C some) there separation is present of myelinated some separation lamella asof a resultmyelinated of high lamella magnification as a result×20,000; of scalehigh magnificationbar = 100 nm that ×20,000; is not scale recognized bar = 100 at nm lower that magnification. is not recognized This at higher lower magnification magnification. delineates This higher the magnificationelectron dense delineates (dense lines—yellow the electron arrows) dense and (dense the electronlines—yellow lucent (intraperiodarrows) and lines—white the electron arrows). lucent (intraperiodM: mitochondria; lines—white N: nucleus arrows) of oligodendrocyte;. M: mitochondria; Nf: neurofilament; N: nucleu OL:s oligodendrocyte.of oligodendrocyte; Nf: neurofilament; OL: oligodendrocyte. The images in the results section may provide additional concepts and improve our understanding The images in the results section may provide additional concepts and improve our of the oligodendrocyte and myelin formation in both the grey matter and transitional zone between understanding of the oligodendrocyte and myelin formation in both the grey matter and transitional the grey matter and the white matter (Figure9 far right—artistic rendition) in the DBC models. zone between the grey matter and the white matter (Figure 9 far right—artistic rendition) in the DBC Indeed, it is obvious that similar to other glial cells, oligodendrocytes respond with cellular and tissue models. Indeed, it is obvious that similar to other glial cells, oligodendrocytes respond with cellular remodeling to the multiple toxicities associated with obesity, insulin resistance, and glucotoxicity and tissue remodeling to the multiple toxicities associated with obesity, insulin resistance, and when the receptor for leptin is deficient as in the DBC model. Of note, OLs not only effect myelination glucotoxicity when the receptor for leptin is deficient as in the DBC model. Of note, OLs not only and their maintenance to individual axons, but also may be important to entire neuronal networks effect myelination and their maintenance to individual axons, but also may be important to entire as a result of shape-shifting—remodeling changes of their ultrastructural morphology including neuronal networks as a result of shape-shifting—remodeling changes of their ultrastructural myelin splitting, ballooning, and axonal collapse. As a result of leptin receptor deficiency, OLs may morphology including myelin splitting, ballooning, and axonal collapse. As a result of leptin receptor remodel their ultrastructure to result in changes of CNS behavioral effects of depression and cognitive deficiency, OLs may remodel their ultrastructure to result in changes of CNS behavioral effects of impairment. Indeed, OLs are dynamic, adaptive, and plastic and are certainly capable of responding depression and cognitive impairment. Indeed, OLs are dynamic, adaptive, and plastic and are via a response to injury mechanism and even aid in the development of abnormal brain behavior in certainly capable of responding via a response to injury mechanism and even aid in the development this diabetic DBC model [11]. of abnormal brain behavior in this diabetic DBC model [11]. The ultrastructural remodeling in the DBC models (Figure5) could represent an early axoplasm The ultrastructural remodeling in the DBC models (Figure 5) could represent an early axoplasm remodeling change of fragmentation of neurofibrillary tangles that are known to be associated with remodeling change of fragmentation of neurofibrillary tangles that are known to be associated with increased possibility of tau pathology in the db/db models of leptin resistance and type 2 diabetes [11]. increased possibility of tau pathology in the db/db models of leptin resistance and type 2 diabetes [11]. Additionally, others have shown similar remodeling changes with fragmentation of axonal neurofilaments in both myelinated and unmyelinated axons similar to what we observed (only in unmyelinated neuronal axons) in streptozotocin induced type 1 diabetic rats [12]. However, the unmyelinated axon we demonstrated (Figure 5) may have depicted some proteinaceous aggregation in DBC models in addition to microtubules.

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Additionally, others have shown similar remodeling changes with fragmentation of axonal neurofilaments in both myelinated and unmyelinated axons similar to what we observed (only in unmyelinated neuronal axons) in streptozotocin induced type 1 diabetic rats [12]. However, the unmyelinated axon we demonstrated (Figure5) may have depicted some proteinaceous aggregation in DBC models in addition to microtubules. In Figures2–4 (cortical grey matter) and Figures6–10 (subcortical white matter transitional zone) we demonstrated splitting, separation, and ballooning of myelinated neurons. These changes have been previously described by others in models of aging [13,14]. Peters et al. state that there are four basic changes in myelin in aged monkey brains, which consist of local splitting, ballooning, redundant myelin, and double myelin sheaths—duplication, which we have depicted in our DBC as compared to the CKC models [13]. These findings seem to also corroborate Parts I and II of this series in that the oligodendrocyte and myelin remodeling changes suggests an accelerated and premature aging process in the diabetic DBC as compared to the control CKC models [14]. In regard to the transitional zone between cortical grey and white matter, the subcortical white matter may represent a region in the brain that is less vascularized [15–17]. Interestingly, our observational findings regarding vascularity in the cortex revealed that there are usually 2–4 neurovascular units (NVU) capillaries per grid examined and in the transitional zone described there was usually only one NVU capillary at the most with a preponderance of no NVU capillaries per grid examined. Additionally, an older paper states the following, “it is possible that the junction between cortex and white matter is relatively avascular, and in this respect this area may be similar to the particular periventricular regions” [18]. Oligodendrocytes and myelination are not static and readily adapt to their environment. The previously described alterations in myelin remodeling in the diabetic DBC models may be detrimental and could impair cognition by the slowing of conduction rates of information—action potential in white matter tracts to distant regions, which could result in a loss of synchronicity not only in individual neurons, but to entire neuronal networks.

4.1. Vascular Stiffness Vascular stiffness including thoracic aorta or carotid artery not only affects the capillaries of the classical end-organs of diabetes (retinopathy, neuropathy, nephropathy, and cardiomyopathy) due to abnormal pulsatile mechanical forces that are associated with microvascular damage and remodeling but may also affect the brain in diabetic cognopathy. In particular, this same cohort that we studied (db/db—DBC) in these three-part series (Parts I, II, and III) has been previously demonstrated to have aortic vascular stiffening [19]. The capillary neurovascular remodeling changes described in Part I could affect the possible diminished microvessels in the transition zones as well as the ones we demonstrated within the grey matter and could decrease the cerebral blood ﬂow to the transitional zone regions as well as the subcortical white matter [20–22]. These NVU remodeling changes could be an additional risk to the development of dysmyelination as we have observed in both the grey matter and transitional zones as described in this paper. The decreased CBF in the TZ and the subcortical white matter (SCWM) could be even more devastating due the possible decreased capillary density found in these regions in comparison to the grey matter [18] (Figure 12). Neuroglia 2018, 1 360 Neuroglia 2018, 1, x FOR PEER REVIEW 10 of 14

Figure 12. Montage of images demonstrating that vascular stiffness in the thoracic or carotid arteries Figure 12. Montage of images demonstrating that vascular stiffness in the thoracic or carotid arteries may result in capillary neurovascular unit remodeling and the possible relationship to the may result in capillary neurovascular unit remodeling and the possible relationship to the development development of dysmyelination in the diabetic DBC models. On the far left are depictions of the of dysmyelination in the diabetic DBC models. On the far left are depictions of the thoracic aorta, thoracic aorta, carotids, and brain circulatory system that are known to experience vascular stiffness carotids, and brain circulatory system that are known to experience vascular stiffness in the diabetic in the diabetic DBC model [19]. The open arrow then points to the fact that there may be neurovascular DBC model [19]. The open arrow then points to the fact that there may be neurovascular uncoupling, uncoupling, which decreases the cerebral blood flow and could result in hypometabolism not only in which decreases the cerebral blood ﬂow and could result in hypometabolism not only in the cortical the cortical grey matter but also in the vulnerable transitional zone. Cerebral flow pulsatility is grey matter but also in the vulnerable transitional zone. Cerebral ﬂow pulsatility is augmented in augmented in type 2 diabetes mellitus (T2DM) [17] and this has been associated with markers of type 2 diabetes mellitus (T2DM) [17] and this has been associated with markers of cerebral damage, cerebral damage, such as white matter hyperintensities in T2DM. Thus, thoracic or carotid artery such as white matter hyperintensities in T2DM. Thus, thoracic or carotid artery stiffness could result stiffness could result in dysmyelination and in turn the dysmyelination could slow the delivery of in dysmyelination and in turn the dysmyelination could slow the delivery of information—action information—action potential without proper synchronicity and result in impaired cognition. potential without proper synchronicity and result in impaired cognition.

Importantly, diabetes is not only a risk factor for stroke, but also a risk factor for the development of whiteImportantly, matter lesions diabetes [23]. is notYotomi only Y. a risket al. factor have for demonstrated stroke, but also that a riskthe db/db factor fordiabetic the development model had greaterof white severity matter lesionsof white [23 ].matter Yotomi damage Y. et al. that have wa demonstrateds associated thatwith the decreased db/db diabetic proliferation model and had survivalgreater severity of oligodendrocyte of white matter progenitor damage cell that [23]. was associated with decreased proliferation and survival of oligodendrocyte progenitor cell [23]. 4.2. Axon Initial Segment Shortening in db/db Diabetic DBC Models 4.2. Axon Initial Segment Shortening in db/db Diabetic DBC Models Keiichiro Susuki’s group has recently demonstrated that diabetic db/db brains (>10 weeks old) Keiichiro Susuki’s group has recently demonstrated that diabetic db/db brains (>10 weeks old) are known to abnormally remodel their neuronal axons, which result in axon initial segment (AIS) are known to abnormally remodel their neuronal axons, which result in axon initial segment (AIS) shortening [24]. While we were unable to demonstrate shortening of the axon initial segment via our shortening [24]. While we were unable to demonstrate shortening of the axon initial segment via our ultrastructural observations, we wish to demonstrate the adjacent nanometer proximity of the ultrastructural observations, we wish to demonstrate the adjacent nanometer proximity of the astrocyte, astrocyte, microglia to the pyramidal neurons. The activation and detachment of the astrocyte and/or microglia to the pyramidal neurons. The activation and detachment of the astrocyte and/or activation activation of microglia provides the potential for cellular crosstalk via ultrastructural astrocyte and of microglia provides the potential for cellular crosstalk via ultrastructural astrocyte and microglia, microglia, which may be a contributing factor for remodeling change in DBC models (Figure 13). which may be a contributing factor for remodeling change in DBC models (Figure 13). Because of the Because of the adjacent nanometer proximity between the glia (astrocytes and microglia) and adjacent nanometer proximity between the glia (astrocytes and microglia) and neurons, we hypothesize neurons, we hypothesize that when microglia are activated as in our DBC model their aberrant leaky that when microglia are activated as in our DBC model their aberrant leaky mitochondria could result mitochondria could result in increased oxidative/nitrosative stress such that the AIS could be in increased oxidative/nitrosative stress such that the AIS could be shortened secondary to excess shortened secondary to excess reactive oxygen/reactive nitrogen species (ROS/RNS) derived from reactive oxygen/reactive nitrogen species (ROS/RNS) derived from activated microglia, intracellular activated microglia, intracellular calcium accumulation, and AIS shortening [24]. Additionally, we calcium accumulation, and AIS shortening [24]. Additionally, we observed abnormalities in myelin observed abnormalities in myelin remodeling—dysmyelination, which could act in synergy with AIS remodeling—dysmyelination, which could act in synergy with AIS shortening and could contribute shortening and could contribute to an increased loss of synchronicity of action potential to distant to an increased loss of synchronicity of action potential to distant sites and result in dysfunctional sites and result in dysfunctional signaling to receiving neurons and neuronal networks. These changes could increase the possibility of impaired cognition as noted in the diabetic db/db DBC models [24–27].

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Neuroglia 2018, 1, x FOR PEER REVIEW 11 of 14 signaling to receiving neurons and neuronal networks. These changes could increase the possibility of impaired cognition as noted in the diabetic db/db DBC models [24–27]. Neuroglia 2018, 1, x FOR PEER REVIEW 11 of 14

FigureFigure 13. Pyramidal 13. Pyramidal cell cell demonstrating demonstrating the the normalnormal axonaxon initial initial segment segment (AIS) (AIS) in ain control a control model. model. Figure 13. Pyramidal cell demonstrating the normal axon initial segment (AIS) in a control model. Note Notethe cytoplasm the cytoplasm of this of this pyramidal pyramidal cell cell has has an an axon hillockhillock region region and and also also the the axon axon initial initial segment segment Note the cytoplasm of this pyramidal cell has an axon hillock region and also the axon initial segment regionregion as the as axonthe axon protrudes protrudes from from the the soma soma ofof thethe pyramidal cell. cell. M: M: myelin; myelin; MGC: MGC: microglial microglial cell; cell; region as the axon protrudes from the soma of the pyramidal cell. M: myelin; MGC: microglial cell; N: pyramidalN: pyramidal nucleus; nucleus; AP AP = acti = actionon potential. potential. Scale Scale bar == 5 5 µm. µm. N: pyramidal nucleus; AP = action potential. Scale bar = 5 µm. Interestingly, we observed ballooning of some axons with evidence of the neuroprotective Interestingly, we observed ballooning of some axons with evidence of the neuroprotective Interestingly,astrocyte being wedetached observed from the ballooning axon and retracted, of some similar axons to with the neurovascu evidencelar of unit the astrocytes neuroprotective in astrocytePart Ibeing of this detached series (Figure from 14)the [5]. axon Astrocytes and retracted, are important similar in to protecting the neurovascu the pyramidallar unit neuronal astrocytes in astrocyte being detached from the axon and retracted, similar to the neurovascular unit astrocytes in Part Iaxons of this in layerseries III (Figure and throughout 14) [5]. Astrocytesthe cortical greyare importantmatter, transitional in protecting zones andthe subcorticalpyramidal white neuronal Part I of this series (Figure 14)[5]. Astrocytes are important in protecting the pyramidal neuronal axons axonsmatter in layer [28]. III and throughout the cortical grey matter, transitional zones and subcortical white inmatter layer [28]. III and throughout the cortical grey matter, transitional zones and subcortical white matter [28].

Figure 14. Detached and retracted astrocytes from a ballooning pyramidal neuronal axon in layer III of cortical grey matter. This is a higher magnification of previous image Figure 4 ×1500. In this image one can observe that there is a region marked with the letter X (yellow) where there is an area of Figuremyelin 14. Detached loss (demyelination) and retracted in astrocytesa ballooned from axon a bawithinllooning layer pyramidal III of the neuronal cortical grey axon matter. in layer III Figure 14. Detached and retracted astrocytes from a ballooning pyramidal neuronal axon in layer of corticalImportantly, grey matter. note the This detachment is a higher and magnification retraction of astrocytes of previous (AC) image from the Figure neuronal 4 ×1500. axon In(defined this image III of cortical grey matter. This is a higher magnification of previous image Figure4 ×1500. In this one canin red observe lettering that and there by dashed is a region lines). Alsomarked note withthe presence the letter of atX least(yellow) three wheraberrante there mitochondria is an area of image one can observe that there is a region marked with the letter X (yellow) where there is an area of myelinin thisloss region. (demyelination) Magnification in×4000; a ballooned scale bar = 0.5axon µm. within layer III of the cortical grey matter. myelin loss (demyelination) in a ballooned axon within layer III of the cortical grey matter. Importantly, Importantly, note the detachment and retraction of astrocytes (AC) from the neuronal axon (defined note theIn summary, detachment we and have retraction attempted of astrocytes to place (AC)this thre frome-part the neuronal series (Parts axon I, (defined II, and inIII) red regarding lettering in red lettering and by dashed lines). Also note the presence of at least three aberrant mitochondria andneurovascular by dashed unit lines). and Also glia note (astrocyte, the presence microglia, of at leastand threeoligodendrocyte) aberrant mitochondria remodeling in changes this region. in in this region. Magnification ×4000; scale bar = 0.5 µm. Magnificationperspective to a× more4000; clinical scale bar setting = 0.5 asµ m.they pertain to type 2 diabetes mellitus in humans (Figure 15).

In summary, we have attempted to place this three-part series (Parts I, II, and III) regarding neurovascular unit and glia (astrocyte, microglia, and oligodendrocyte) remodeling changes in perspective to a more clinical setting as they pertain to type 2 diabetes mellitus in humans (Figure 15).

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In summary, we have attempted to place this three-part series (Parts I, II, and III) regarding neurovascular unit and glia (astrocyte, microglia, and oligodendrocyte) remodeling changes in

Neurogliaperspective 2018, to1, x a FOR more PEER clinical REVIEW setting as they pertain to type 2 diabetes mellitus in humans (Figure12 of 15 14).

Figure 15. Multiple predisposing risk factors and metabolic and structural defects in DBC models. Figure 15. Multiple predisposing risk factors and metabolic and structural defects in DBC models. This figure depicts the multiple predisposing risk factors, metabolic and our ultrastructural This figure depicts the multiple predisposing risk factors, metabolic and our ultrastructural remodeling remodeling that we have shared regarding the neurovascular unit and glia including the astrocyte that we have shared regarding the neurovascular unit and glia including the astrocyte (AC), microglia, (AC), microglia, and oligodendrocyte in this three-part series. The blue ovals at the bottom of the and oligodendrocyte in this three-part series. The blue ovals at the bottom of the image depict the image depict the multiple situations that might give rise to the remodeling changes and how they are multiple situations that might give rise to the remodeling changes and how they are related to each of related to each of the three-part series as Part I, Part II, and Part III publications as follows: Aging, the three-part series as Part I, Part II, and Part III publications as follows: Aging, lifestyle, environment, lifestyle, environment, genetics—particularly the leptin receptor deficiencies in the DBC and also the genetics—particularly the leptin receptor deficiencies in the DBC and also the numerous single numerous single nucleotide polymorphisms (SNPs) in humans, and comorbidities associated with nucleotide polymorphisms (SNPs) in humans, and comorbidities associated with obesity (metabolic obesity (metabolic syndrome and accelerated atherosclerosis). a.k.a: also known as; AD: syndrome and accelerated atherosclerosis). a.k.a: also known as; AD: Alzheimer’s disease; aMGC: Alzheimer’sactivated microglia disease; cell; aMGC: BBB: blood-brainactivated microglia barrier; db/db:cell; BBB: DBC; blood-brain HTN: hypertension; barrier; db/db: IL-1β :DBC; interleukin HTN: 1hypertension; beta; IR: insulin IL-1 resistance;β: interleukin NVU: 1 neurovascular beta; IR: insulin unit; OL:resistance; oligodendrocyte; NVU: neurovascular PD: Parkinson’s unit; disease; OL: oligodendrocyte;PKCβ: protein kinase PD: CPa beta;rkinson’s R: receptor; disease; ROS/RNS: PKCβ: reactiveprotein oxygen/reactivekinase C beta; nitrogenR: receptor; species; ROS/RNS: SCWM: reactivesubcortical oxygen/reactive white matter; nitrogen T2DM: typespecies; 2 diabetes SCWM: mellitus; subcortical TGF whiteβ: transforming matter; T2DM: growth type factor2 diabetes beta; mellitus;TNFα: tumor TGFβ necrosis: transforming factor alpha; growth TZ: factor transition beta; zone.TNFα: tumor necrosis factor alpha; TZ: transition zone. 5. Future Directions 5. Future Directions Although we have not yet been able to study oligodendrocyte and myelin extensively with Although we have not yet been able to study oligodendrocyte and myelin extensively with focused ion beam/scanning electron microscopy technology as we have previously done with the focused ion beam/scanning electron microscopy technology as we have previously done with the microglia in supplement videos in Part II of this series [29], we look forward to this possibility in due microglia in supplement videos in Part II of this series [29], we look forward to this possibility in due time such that we may be able to better understand their 3D stacks to create oligodendrocyte and time such that we may be able to better understand their 3D stacks to create oligodendrocyte and myelin videos. Additionally, we hope to study oligodendrocytes and myelin remodeling specifically in myelin videos. Additionally, we hope to study oligodendrocytes and myelin remodeling specifically the corpus callosum and/or optic nerves and how they relate to elongated white matter tracts. Indeed, in the corpus callosum and/or optic nerves and how they relate to elongated white matter tracts. these are exciting times to study the ultrastructural remodeling changes in the glia. Indeed, these are exciting times to study the ultrastructural remodeling changes in the glia. Author Contributions: M.R.H. and V.G.D. conceptualized the study; M.R.H. performed the image collection and Authorinterpretation; Contributions: D.G.G. prepared M.R.H. and the tissueV.G.D. specimens conceptualized for transmission the study; electron M.R.H. microscopicperformed the studies image and collection assisted andM.R.H.; interpretation; M.R.H. prepared D.G.G.the prepared manuscript; the tissue A.A. specimens collected tissue for transmission specimens; V.G.D.,electron A.A., microscopic and D.G.G. studies assisted and assistedin editing. M.R.H.; M.R.H. prepared the manuscript; A.A. collected tissue specimens; V.G.D., A.A., and D.G.G. assistedFunding: inThis editing. study was supported by an internal grant entitled Excellence in Electron Microscopy grant and issued by the University of Missouri Electron Microscopy Core and Office of Research. No external funding Funding:was available. This study was supported by an internal grant entitled Excellence in Electron Microscopy grant and issued by the University of Missouri Electron Microscopy Core and Office of Research. No external funding was available.

Acknowledgments: Authors wish to thank Tommi White of the University of Missouri Electron Microscopy Core Facility. Authors also wish to thank our mentor James R. Sowers, who inspired us to interrogate, characterize, and understand the secrets that the tissues behold.

Conflicts of Interest: The authors declare no conflict of interest.

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References

1. Michalski, J.P.; Kothary, R. Oligodendrocytes in a nutshell. Front. Cell. Neurosci. 2015, 9, 340. [CrossRef] [PubMed] 2. Peters, A.; Palay, S.L.; Webster, H. The Fine Structure of the Nervous System—Neurons and Their Supportive Cells—The Cellular Sheaths of Neurons, 3rd ed.; Oxford University Press: New York, NY, USA, 1991; pp. 212–261. ISBN 0-19-506571-9. 3. Pukos, N.; Yoseph, R.; McTigue, D.M. To Be or Not to Be: Environmental Factors that Drive Myelin Formation during Development and after CNS Trauma. Neuroglia 2018, 1, 63–90. [CrossRef] 4. Boullerne, A.I. The history of myelin. Exp. Neurol. 2016, 283, 431–445. [CrossRef][PubMed] 5. Hayden, M.R.; Grant, D.; Aroor, A.; Demarco, V.G. Ultrastructural Remodeling of The Neurovascular Unit in The Female Diabetic db/db Model—Part I: Astrocyte. Neuroglia 2018, 1, 220–244. [CrossRef] 6. Montagne, A.; Nikolakopoulou, A.M.; Zhao, Z.; Sagare, A.P.; Si, G.; Lazic, D.; Barnes, S.R.; Daianu, M.; Ramanathan, A.; Go, A.; et al. Pericyte degeneration causes white matter dysfunction in the mouse central nervous system. Nat. Med. 2018, 24, 326–337. [CrossRef][PubMed] 7. Reijmer, Y.D.; Brundel, M.; de Bresser, J.; Kappelle, L.J.; Leemans, A.; Biessels, G.J. Utrecht Vascular Cognitive Impairment Study Group. Microstructural white matter abnormalities and cognitive functioning in type 2 diabetes: A diffusion tensor imaging study. Diabetes Care 2013, 36, 137–144. [CrossRef][PubMed] 8. Cavaglia, M.; Dombrowski, S.M.; Drazba, J.; Vasanji, A.; Bokesch, P.M.; Janigro, D. Regional variation in brain capillary density and vascular response to ischemia. Br. Res. 2001, 910, 81–93. [CrossRef] 9. Walhovd, K.B.; Johansen-Berg, H.; Káradóttir, R.T. Unraveling the secrets of white matter—Bridging the gap between cellular, animal and human imaging studies. Neuroscience 2014, 276, 2–13. [CrossRef][PubMed] 10. Snaidero, N.; Möbius, W.; Czopka, T.; Hekking, L.H.; Mathisen, C.; Verkleij, D.; Goebbels, S.; Edgar, J.; Merkler, D.; Lyons, D.A.; et al. Myelin membrane wrapping of CNS axons by PI(3,4,5)P3-dependent polarized growth at the inner tongue. Cell 2014, 156, 277–290. [CrossRef][PubMed] 11. Platt, T.L.; Beckett, T.L.; Kohler, K.; Niedowicz, D.M.; Murphy, M.P. Obesity, diabetes, and leptin resistance promote tau pathology in a mouse model of disease. Neuroscience 2016, 315, 162–174. [CrossRef][PubMed] 12. Hernández-Fonseca, J.P.; Rincón, J.; Pedreañez, A.; Viera, N.; Arcaya, J.L.; Carrizo, E.; Mosquera, J. Structural and ultrastructural analysis of cerebral cortex, cerebellum, and hypothalamus from diabetic rats. Exp. Diabetes Res. 2009, 2009, 329632. [CrossRef][PubMed] 13. Peters, A.; Moss, M.B.; Sethares, C. Effects of aging on myelinated nerve fibers in monkey primary visual cortex. J. Comp. Neurol. 2000, 419, 364–376. [CrossRef] 14. Wrighten, S.A.; Piroli, G.G.; Grillo, C.A.; Reagan, L.P. A look inside the diabetic brain: Contributors to diabetes-induced brain aging. Biochim. Biophys. Acta 2009, 1792, 444–453. [CrossRef][PubMed] 15. Bowley, M.P.; Cabral, H.; Rosene, D.L.; Peters, A. Age changes in myelinated nerve fibers of the cingulate bundle and corpus callosum in the rhesus monkey. J. Comp. Neurol. 2010, 518, 3046–3064. [CrossRef][PubMed] 16. Sosa, S.M.; Smith, K.J. Understanding a role for hypoxia in lesion formation and location in the deep and periventricular white matter in small vessel disease and multiple sclerosis. Clin. Sci. 2017, 131, 2503–2524. [CrossRef][PubMed] 17. Nonaka, H.; Akima, M.; Hatori, T.; Nagayama, T.; Zhang, Z.; Ihara, F. Microvasculature of the human cerebral white matter: Arteries of the deep white matter. Neuropathology 2003, 23, 111–118. [CrossRef][PubMed] 18. Brownell, B.; Hughes, J.T. The distribution of plaques in the cerebrum in multiple sclerosis. J. Neurol. Neurosurg. Psychiatr. 1962, 25, 315–320. [CrossRef] 19. Aroor, A.R.; Das, N.A.; Carpenter, A.J.; Habibi, J.; Jia, G.; Ramirez-Perez, F.I.; Martinez-Lemus, L.; Manrique-Acevedo, C.M.; Hayden, M.R.; Duta, C.; et al. Glycemic control by the SGLT2 inhibitor empagliflozin decreases aortic stiffness, renal resistivity index and kidney injury. Cardiovasc. Diabet. 2018, 17, 108. [CrossRef][PubMed] Neuroglia 2018, 1 364

20. Cooper, L.L.; Mitchell, G.F. Aortic Stiffness, Cerebrovascular Dysfunction, and Memory. Pulse 2016, 4, 69–77. [CrossRef][PubMed] 21. Heffernan, K.S. Carotid artery stiffness and cognitive function in adults with and without type 2 diabetes: Extracranial contribution to an intracranial problem? Atherosclerosis 2016, 253, 268–269. [CrossRef][PubMed] 22. Lee, K.O.; Lee, K.Y.; Lee, S.Y.; Ahn, C.W.; Park, J.S. Lacunar infarction in type 2 diabetes is associated with an elevated intracranial arterial pulsatility index. Yonsei Med. J. 2007, 48, 802–806. [CrossRef][PubMed] 23. Yatomi, Y.; Tanaka, R.; Shimada, Y.; Yamashiro, K.; Liu, M.; Mitome-Mishima, Y.; Miyamoto, N.; Ueno, Y.; Urabe, T.; Hattori, N. Type 2 diabetes reduces the proliferation and survival of oligodendrocyte progenitor cells in ischemia white matter lesions. Neuroscience 2015, 289, 214–223. [CrossRef][PubMed] 24. Yermakov, L.M.; Drouet, D.E.; Griggs, R.B.; Khalid, M.; Elased, K.M.; Susuki, K. Type 2 Diabetes Leads to Axon Initial Segment Shortening in db/db Mice. Front. Cell. Neurosci. 2018, 12, 146. [CrossRef][PubMed] 25. Benusa, S.D.; George, N.M.; Sword, B.A.; DeVries, G.H.; Dupree, J.L. Acute neuroinﬂammation induces AIS structural plasticity in a NOX2-dependent manner. J. Neuroinﬂamm. 2017, 14, 116. [CrossRef][PubMed] 26. Rasband, M.N. The axon initial segment and the maintenance of neuronal polarity. Nat. Rev. Neurosci. 2010, 11, 552–562. [CrossRef][PubMed] 27. Bender, K.J.; Trussell, L.O. The physiology of the axon initial segment. Annu. Rev. Neurosci. 2012, 35, 249–265. [CrossRef][PubMed] 28.B élanger, M.; Magistretti, P.J. The role of astroglia in neuroprotection. Dialog. Clin. Neurosci. 2009, 11, 281–295. 29. Hayden, M.R.; Grant, D.; Aroor, A.; Demarco, V.G. Ultrastructural Remodeling of The Neurovascular Unit in The Female Diabetic db/db Model—Part II: Microglia and Mitochondria. Neuroglia 2018, 1, 311–326. [CrossRef]

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