Aging causes morphological alterations in astrocytes and microglia in human substantia nigra pars compacta

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Aging causes morphological alterations in astrocytes and microglia in human substantia nigra pars compacta
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  Aging causes morphological alterations in astrocytes and microgliain human substantia nigra pars compacta H.J. Jyothi a , D.J. Vidyadhara a , Anita Mahadevan b , Mariamma Philip c ,Suresh Kumar Parmar b , S. Gowri Manohari a , S.K. Shankar b , Trichur R. Raju a ,Phalguni Anand Alladi a , * a Department of Neurophysiology, National Institute of Mental Health and Neuro Sciences, Bengaluru, India b Department of Neuropathology, National Institute of Mental Health and Neuro Sciences, Bengaluru, India c Department of Biostatistics, National Institute of Mental Health and Neuro Sciences, Bengaluru, India a r t i c l e i n f o  Article history: Received 28 April 2015Received in revised form 30 July 2015Accepted 25 August 2015 Keywords: Parkinson ’ s diseaseAutopsied human midbrainsStereologyMorphometryDensitometryGFAPCNPaseIba1S100 b a b s t r a c t Age being a risk factor for Parkinson ’ s disease, assessment of age-related changes in the human sub-stantia nigra may elucidate its pathogenesis. Increase in Marinesco bodies,  a -synuclein, free radicals andso forth in the aging nigral neurons are clear indicators of neurodegeneration. Here, we report the glialresponses in aging human nigra. The glial numbers were determined on Nissl-stained sections. Theexpression of glial  fi brillary acidic protein, S100 b , 2 0 , 3 0 -cyclic nucleotide 3 0 phosphodiesterase, and Iba1was assessed on cryosections of autopsied midbrains by immunohistochemistry and densitometry. Theglial counts showed a biphasic increase, of which, the  fi rst prominent phase from fetal age to birth couldbe physiological gliogenesis whereas the second one after middle age may re fl ect mild age-relatedgliosis. Astrocytic morphology was altered, but glial  fi brillary acidic protein expression increased onlymildly. Presence of type-4 microglia suggests possibility of neuroin fl ammation. Mild reduction in 2 0 , 3 0 -cyclic nucleotide 3 0 phosphodiesterase e labeled area denotes subtle demyelination. Stable age-relatedS100 b  expression indicates absence of calcium overload. Against the expected prominent gliosis, sub-tle age-related morphological alterations in human nigral glia attribute them a participatory role inaging.   2015 Elsevier Inc. All rights reserved. 1. Introduction Aging is a major risk factor for Parkinson ’ s disease (PD). Bothaging and PD display overlapping neuroanatomical changes in themidbrain(Collieretal.,2011).Themorphologicalalterationsseeninaging brain are steered bygenetic composition of the individuals aswell as environmental conditions. Both aging and neurodegenera-tive disorders share commonalities such as neuronal loss, gliosis,loss of myelination (Peters, 2002), disruption of calcium homeo-stasis (Nixon, 2003), and so forth. Other features such as neuro- in fl ammation (Sherer et al., 2003), oxidative stress, mitochondrial dysfunction,  a -synuclein aggregation, and alteration in proteindegradation pathways are reported in PD and aging (Hirsch et al.,2012). These are temporal yet stochastic events arbitrated by glia(Kanaan et al., 2010). Studies thus far acknowledge that astroglial, microglial, and oligodendrocytic populations mayundergochangesas a consequence of compensatory mechanisms or degenerativecues of aging and neurodegenerative disorders.Several brain regions show variable but de fi nite age-relatedchanges in glial  fi brillary acidic protein (GFAP) expression and itsmessenger RNA levels in astrocytes, suggesting age-associatedastrogliosis (Goss et al., 1991). Contrary to this, in the nonhumanprimate substantia nigra, the number of GFAP immunoreactive gliaincreased neither in response to 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) injection (Kanaan et al., 2008) norduring normal aging (Kanaan et al., 2010). Astrocytic activationwastherefore not considered to be a contributory factor in MPTP-induced degeneration and normal aging-related regional vulnera-bility. However, few others reported remarkable changes inastroglial and microglial structure after MPTP intoxication in theseprimates(Barcia etal., 2009). Furthermore,con fl ictingobservationshave been inferred from GFAP expression studies in humans in PD.Forexample,nosigni fi cantdifferenceinGFAPstainingwasnotedinsubstantianigrabetweenPDcases andcontrols(Banatiet al.,1998).In another study, a direct correlation between levels of GFAP anddisease progression was reported in the hypothalamus of PDpatients (Thannickal et al., 2007). There are no reports on the  Jyothi H. J. and Vidyadhara D. J. contributed equally. *  Corresponding author at: Department of Neurophysiology, National Institute of Mental Health and Neuro Sciences, Hosur Road, Bengaluru 560029, India. Tel.: 918026995179; fax: 91 80 26562121. E-mail address:  alladiphalguni@gmail.com (P.A. Alladi). Contents lists available at ScienceDirect Neurobiology of Aging journal homepage: www.elsevier.com/locate/neuaging 0197-4580/$  e  see front matter    2015 Elsevier Inc. All rights reserved.http://dx.doi.org/10.1016/j.neurobiolaging.2015.08.024 Neurobiology of Aging xxx (2015) 1 e 13  astrocytic response to aging in human substantia nigra pars com-pacta (SNpc).Microglia are the cells of mesohematopoietic srcin which pro-tectthebrainenvironmentbyinitiatingaquickresponsetochangesand effectively modulate in fl ammation. They can be morphologi-cally typed into rami fi ed (resting microglia), intermediate, acti-vated, and amoeboid or phagocytic microglia. Rami fi ed microgliahave a small oval soma with numerous and branched processes.These processes help in maintaining brain homeostasis undernormal condition. In fl ammatory signals transform the rami fi edones into activated and phagocytic microglia (Barcia et al., 2011;Kanaan et al., 2008). During early postnatal development in mice,the amoeboid microglial cells phagocytose neurons undergoingprogrammed cell death (Schafer et al., 2012). Occurrence of age- related, nigra-associated microgliosis was reported in Rhesusmonkeys (Kanaan et al., 2010). Signi fi cant increase in microglialreactivity and in fl ammatory markers were seen in the ventralmidbrain of PD patients (Imamura et al., 2003). A major RNAse enzyme of myelin, 2 0 , 3 0 -cyclic nucleotide 3 0 phosphodiesterase (CNPase) is bound to the oligodendrocyticmembrane. It is found speci fi cally in oligodendrocytes andSchwann cells and makes up to 4% of the total central nervoussystem myelin proteins and is considered to be an early marker foroligodendrocytes (KuriharaandTsukada,1967).Itispresenteveninthe noncompact form of myelin (Trapp et al., 1988). CNPase-de fi cient mice show delayed onset of neurogenesis and also theoccurrence of periaxonal gliosis, the latter leads to neuro-degeneration. Thus, CNPase is involved in the maintenance of axonal structure (Rasband et al., 2005). The myelin membrane degradation occurs in the presence of micromolar calcium con-centration which activates calpain-1 and is enhanced with aging(Sloane et al., 2003). Status of CNPase expression or response of oligodendrocytes in aging human SNpc is not known.Ca 2 þ in fl ux is a primary event in several processes associatedwith neuronal activity, and balancing its intracellular and extra-cellularconcentrationattainsenormoussigni fi cance(Barres,2008). Astrocytes present in close proximity to the neurons quench theCa 2 þ throughvariousmechanismstostabilizeittothephysiologicallevel. In situations of low extracellular Ca 2 þ , glial cells maintain thehomeostasis and thus, represent regulatory role (Blaustein et al.,1996; Zanotti and Charles, 1997). In normally aging mice nigra, the neuronal membrane capacitance is unaffected; however, the l-type calcium currents are smaller and hence the related channelsmay be compromised (Branch et al., 2014). Yet, whether the gliacompensate for the calcium imbalance or not is not understood.S100 b  is a glial calcium-binding protein, the expression of which ishigh in autopsied brains of PD patients and in MPTP-injected mice(Sathe et al., 2012). Understanding the expression pattern in aging nigra may further elucidate its role in the PD pathogenesis.Although few studies report notable nigral neuronal death withage (Chu et al., 2002; Ma et al.,1999), others report absence of such age-related cell loss (Alladi et al., 2009; Kubis et al., 2000). Sys- tematic studies on the effects of aging on glial proteins in humansubstantia nigra are sparse. Because glia play a vital role in thesurvival or loss of neurons and they modulate age-related neuro-degeneration in the SNpc, in the present study we evaluated thechanges in the expression of glial proteins with age. 2. Materials and methods  2.1. Tissue collection and processing  The archival brain tissues of Asian Indians were collected atautopsy by pathologists at the Human Brain Tissue Repository,NIMHANS, Bangaluru (number of specimens  ¼  36; age range 28weeks of gestation [GW] e 88 years). The subjects were neurologi-cally normal individuals who succumbed to road traf  fi c accidents.The fetal specimen was that from a medically terminated preg-nancy at 28 GW. The postmortem interval ranged from 40 minutesto 25 hours. The hemisected midbrains were sliced and immersion fi xed in chilled 4% paraformaldehyde for 48 hours, followed bycryoprotection in sucrose gradients of 15% and 30% and sectionedcoronally(Leica CM 1510 S) into 40 m  thick serial sections (Chu et al.,2002;Kanaanetal.,2008,2010)andcollectedonsubbedslides.The study was approved by the Institutional Human Ethics Committee.  2.2. Cresyl violet staining  Every20thsectionwasrinsedinchloroformandstainedwith1%buffered cresyl violet for 5 minutes at 60   C. The sections weredifferentiatedanddehydratedinincreasingalcoholgradesfollowedby clearing in xylene. The slides were air-dried and coverslippedusing xylene and distyrene plasticizer xylol (Alladi et al., 2009).  2.3. Stereological quanti  fi cation of glia on Nissl-stained sections We made a minor modi fi cation to our earlier protocol to quan-tify the numberof glia on Nissl-stained sections. As before,weusedtheopticalfractionatorsamplingdesignonevery20thsection,thus,we studied approximately 10 sections per coded specimen (Alladiet al., 2009). Within the anatomical boundaries of substantianigra (Halliday et al., 2005; Siddiqi and Peters,1999), the glia were quanti fi ed using 100   objective of the Olympus BX61 Microscope(Olympus Microscopes Inc, Japan) equipped with Stereo Investi-gator software version 7.2 (Microbright fi eld Inc, Colchester, USA).The sampling parameters such as probe size (45  m m  45  m m) andgridsize(450 m m  450 m m)wereslightmodi fi cationsofourearlierprotocol for quanti fi cation of neurons in the same area. Thesemodi fi cations were made due to the differences in size of theneurons and glia. The absolute numbers were computed using the “ .DAT ”  fi les of optical fractionator probe (Alladi et al., 2009).  2.4. Immunohistochemistry procedure The immunoperoxidase labeling protocol was identical to thatreported earlier (Alladi et al., 2010a). Separate series of sectionswere used for immunolabeling each of the glial proteins namelyGFAP (1:1500, mouse monoclonal, ICN Chemical Inc, USA), S100 b (1:500, mouse monoclonal, Sigma-Aldrich, USA), CNPase (1:1500,mouse monoclonal, Abcam, USA), and Iba1 (1:500, mouse mono-clonal, Abcam). Because microglia can be effectively labeled inhuman brains using an antibody against Iba1, we considered usingthe same marker (Ahmed et al., 2007; Nguyen et al., 2013). All the primaries were raised in mouse; hence goat anti-mouse antibodywas the preferred secondary antibody (1:200 dilution; VectorLaboratories, Burlingame, USA). This was followed by tertiary la-beling with avidin-biotin complex (1:200, Elite ABC kits; VectorLaboratories). We visualized the staining using 0.05% 3 0 -3 0 -dia-minobenzidine(DAB)and0.03%H 2 O 2 asachromogen.We fi xedtheDAB reaction time to 60 seconds after addition of the chromogensolution because at 70 second the proportion of brown precipitatesobtained by peroxidase-catalyzed reaction reached a plateau. Thisprotocol is being followed routinely in our laboratory (Alladi et al.,2009, 2010a). The sections were counterstained with 1% buffered cresyl violetfollowed by differentiation and dehydration in increasing alcoholgrades followed byclearing in xylene.The slides wereair-dried andcoverslipped using xylene and distyrene plasticizer xylol (Alladiet al., 2009; Beach et al., 2007; Chu et al., 2002). H.J. Jyothi et al. / Neurobiology of Aging xxx (2015) 1 e 13 2   2.5. Subdivisions of nigra To decipher the impact of glia on neuronal loss and in estab-lishing regional vulnerability within the nigra, we divided the nigrainto 3 parts based on the number of nigrosomes present. Nigro-somes,asde fi nedearlier,arecellclusterswithinthenigrawithpoorcalbindin expression, which renders themvulnerable to death. Fivenigrosomes have been described to exist in the substantia nigra(Damier et al.,1999). In the present study, the regions were termed as medial, intermediate, and ventrolateral sections and/or sub-regions. The medial nigra, as the name suggests is located medially,but is distinct from the ventral tegmental area and contains thenigrosome 2. The ventrolateral nigra contains 4 major nigrosomes,that is, 1, 3, 4, and 5 and hence is maximally vulnerable. The in-termediate nigra is the band between the medial and ventrolateralsections which does not contain any nigrosome. Fig.1.  Age-related gliosis in human substantia nigra pars compacta (SNpc). Photomicrographs of Nissl-stained human SNpc from different age groups: (A) 28 weeks of gestation, (B)1 year, (C) 18 years, (D) 35 years, (E) 50 years, (F) 88 years. Note that the neurons contain neuromelaninpigment and are larger (A and B, arrows) than the glia (A and B, arrowheads).The scatter plot (G) shows an increase in the number of glia with age. The histogram (H) of different age groups shows that the increase is more prominent from fetal to young agegroup. Scale bar (A e F) 50  m m.  Table 1 Number of Nissl-stained glia in human SNpc in different decadesAge groups (y) Number of glia  standard deviation Fold changeFetal 7.67  0  10 5 d 1 e 10 14.3  1.4  10 5 1.8711 e 19 15.0  2.9  10 5 0.9620 e 29 12.6  1.3  10 5 0.8430 e 39 15.1  2.4  10 5 1.2040 e 49 14.7  2.8  10 5 0.9850 e 59 17.5  0  10 5 1.1960 e 69 11.4  1.0  10 5 0.6570 e 79 16.9  1.3  10 5 1.4980 e 89 17.0  1.2  10 5 0.99Note almost 2-fold change in the number of glia from the fetal age to fi rst decade. Inthe subsequent decades, there were no signi fi cant changes. A value of more than 1fold change indicates an increase.Key: SNpc, substantia nigra pars compacta. H.J. Jyothi et al. / Neurobiology of Aging xxx (2015) 1 e 13  3   2.6. Densitometry-based image analysis Densitometric image analysis to measure staining intensity wasperformed on high magni fi cation images captured with 40  objective using a  “ Windows ”  based image analysis system (Q WinV3, Leica Systems, Germany) as per our earlier protocol. The in-tensity output was valued on gray scale of 0 e 255, where 0 equalsintense staining and 255 means absence of staining. Thus,lower gray values suggest higher protein expression and vice versa(Alladi et al., 2010a). The  “ measure  fi eld ”  command was thenapplied to quantify the number of pixels or the  “ area stained ” .  2.7. Soma size measurement and morphological typing  The soma size measurement (Alladi et al., 2009) as well asmorphological typing of the GFAP and Iba1-labeled glia (Kanaanet al., 2010) was performed with a  “ Windows ”  based imageanalysis system (Q Win V3, Leica Systems). Approximately, 750 Fig. 2.  Age-related alterations in glial  fi brillary acidic protein (GFAP) expression. Photomicrographs of GFAP-stained human substantia nigra pars compacta representing differentage groups: (A) 1 year, (B) 4 years, (C) 18 years, (D) 35 years, (E) 50 years, (F) 80 years. Note that the area stained with GFAP increases slightly with age (G), whereas the intensityremains comparable (H). Scale bar (A e F) 25  m m. H.J. Jyothi et al. / Neurobiology of Aging xxx (2015) 1 e 13 4  GFAP-labeled astroglia per specimen were analyzed. Brie fl y, theastroglia were classi fi ed as resting or type 1, that is, the cells withlight cytoplasmic staining and slender processes, type 2 or inter-mediate reactive astrocytes which had more intense GFAP labeling,larger soma, and slightly thick processes, or type 3 which werehypertrophied and had much larger soma and highly intense GFAPexpression.Similarly, microglial typing was performed on approximately350 Iba1-labeled cells per specimen, based on their size and pro-cesses. Brie fl y, the resting or type 1 were the cells with light cyto-plasmic staining and branched processes, type 2 or intermediatereactive microglia had more intense labeling, larger soma, and borethicker processes, type 3 were yet larger and had highly intenseIba1 expression, whereas the type 4 or amoeboid microglia werealmost globose and bore no processes.  2.8. Statistical analysis We applied the Pearson ’ s product moment correlation coef  fi -cient to analyze the changes in the mean number of Nissl-stainedglia and relative expression of the glial proteins, namely GFAP,S100 b , CNPase, and Iba1 measured by densitometric image analysisin relation to increasing age. The statistics were performed usingthe SPSS software version 22. Appropriate post hoc analysis was  Table 2 Fold change in the area and intensity of GFAP expression in human SNpcAge groups(y)GFAP expressionArea ( m m 2 ) Fold change Intensity Fold change0.28 1.66  10 5 d  122.80  d 1 e 10 1.40  10 5 0.84 137.82 1.1211 e 19 1.87  10 5 1.33 127.23 0.9220 e 29 2.55  10 5 1.36 103.93 0.8230 e 39 2.18  10 5 0.85 114.77 1.1040 e 49 1.81  10 5 0.83 114.10 0.9950 e 59 2.10  10 5 1.16 104.70 0.9260 e 69 1.80  10 5 0.86 132.85 1.2770 e 79 2.18  10 5 1.21 110.75 0.8380 e 89 2.00  10 5 0.92 135.78 1.23There werenostatistically signi fi cantage-related changesin botharea and intensityof GFAP expression. The intensity output was valued on gray scale of 0 e 255, where0equals intense staining and 255means absence of staining.Thus lower gray valuessuggest higher protein expression and vice versa. With respect to the fold changes,values higher than 1 suggest an increase.Key: GFAP, glial  fi brillary acidic protein; SNpc, substantia nigra pars compacta. Fig. 3.  Aging causes morphological alterations in astrocytes. Photomicrographs of different morphological types of astrocytes in the human substantia nigra pars compacta,identi fi ed by glial  fi brillary acidic protein immunostaining. (A) The type 1 astrocytes have long slender processes (arrows) and sparsely-stained cytoplasm (asterisk). (B) The type 2astrocytes have relatively thicker processes (arrows) and intensely-stained soma (asterisk). (C) The type 3 astrocytes have intensely-stained cytoplasm (asterisk), the nucleus isbarely visible, and the processes (arrows) are thick and stubby. A difference in soma size was perceived such that type 1 < type 2 < type 3 astrocytes. Scale bar (A e C) 5  m m. Note thesmall dip in type 1 astrocytes whereas the type 2 astrocytes showed a mild increase in their numbers, although not statistically signi fi cant (D, scatter plot, dashed trendline). A mildincrease in the soma size was seen in the intermediate (IM) and ventrolateral (VL) regions (E, scatter plot). Abbreviation: M, medial.  Table 3 Age-related alterations in GFAP immunoreactive astrocytic numbers and soma size,in human SNpcAge groups (y) AstrocytesNumberper  fi eldFoldchangeMean somaarea ( m m 2 )Foldchange0.28 36  d  1026  d 1 e 10 37 1.03 1158 1.1311 e 19 37 1.00 1313 1.1320 e 29 43 1.16 1367 1.0430 e 39 22 0.51 1812 1.3340 e 49 35 1.60 1512 0.8350 e 59 36 1.03 1709 1.1360 e 69 41 1.14 1130 0.6670 e 79 45 1.10 1278 1.1380 e 89 46 1.02 1386 1.09The average number of astrocytes remained comparable at all the stages studied.There was a trend toward increase in soma size, which indicates the phenotypicswitch over with aging. With respect to the fold changes, values higher than 1suggest an increase.Key: GFAP, glial  fi brillary acidic protein; SNpc, substantia nigra pars compacta. H.J. Jyothi et al. / Neurobiology of Aging xxx (2015) 1 e 13  5
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