cannula-implantation-into-the-cisterna-magna-of-rodents

Cannula Implantation into the Cisterna Magna of Rodents

Cisterna magna cannulation (CMc) is a straightforward procedure that enables direct access to the cerebrospinal fluid (CSF) without operative damage to ...

University of Rochester Medical Center

Hypersynchronous neuronal firing is a hallmark of epilepsy, but the mechanisms underlying simultaneous activation of multiple neurons remains unknown. Epileptic discharges are in part initiated by a local depolarization shift that drives groups of neurons into synchronous bursting. In an attempt to define the cellular basis for hypersynchronous bursting activity, we studied the occurrence of paroxysmal depolarization shifts after suppressing synaptic activity using tetrodotoxin (TTX) and voltage-gated Ca(2+) channel blockers. Here we report that paroxysmal depolarization shifts can be initiated by release of glutamate from extrasynaptic sources or by photolysis of caged Ca(2+) in astrocytes. Two-photon imaging of live exposed cortex showed that several antiepileptic agents, including valproate, gabapentin and phenytoin, reduced the ability of astrocytes to transmit Ca(2+) signaling. Our results show an unanticipated key role for astrocytes in seizure activity. As such, these findings identify astrocytes as a proximal target for the treatment of epileptic disorders.

An astrocytic basis of epilepsy.

Although astrocytes are the most abundant cell type in the brain, evidence for their activation during physiological sensory activity is lacking. Here we show that whisker stimulation evokes increases in astrocytic cytosolic calcium (Ca(2+)) within the barrel cortex of adult mice. Increases in astrocytic Ca(2+) were a function of the frequency of stimulation, occurred within several seconds and were inhibited by metabotropic glutamate receptor antagonists. To distinguish between synaptic input and output, local synaptic activity in cortical layer 2 was silenced by iontophoresis of AMPA and NMDA receptor antagonists. The antagonists did not reduce astrocytic Ca(2+) responses despite a marked reduction in excitatory postsynaptic currents in response to whisker stimulation. These findings indicate that astrocytes respond to synaptic input, by means of spillover or ectopic release of glutamate, and that increases in astrocytic Ca(2+) occur independently of postsynaptic excitatory activity.

Astrocytic Ca2+ signaling evoked by sensory stimulation in vivo.

Cortical spreading depression (CSD) is a self-propagating wave of cellular depolarization that has been implicated in migraine and in progressive neuronal injury after stroke and head trauma. Using two-photon microscopic NADH imaging and oxygen sensor microelectrodes in live mouse cortex, we find that CSD is linked to severe hypoxia and marked neuronal swelling that can last up to several minutes. Changes in dendritic structures and loss of spines during CSD are comparable to those during anoxic depolarization. Increasing O2 availability shortens the duration of CSD and improves local redox state. Our results indicate that tissue hypoxia associated with CSD is caused by a transient increase in O2 demand exceeding vascular O2 supply.

Cortical spreading depression causes and coincides with tissue hypoxia.

&#x76AE;&#x8D28;&#x6269;&#x6563;&#x6027;&#x6291;&#x5236; (CSD) &#x662F;&#x6709;&#x7275;&#x8FDE;&#x5728;&#x504F;&#x5934;&#x75DB;&#x548C;&#x8111;&#x4E2D;&#x98CE;&#x548C;&#x8111;&#x5916;&#x4F24;&#x540E;&#x9010;&#x6B65;&#x795E;&#x7ECF;&#x7EC6;&#x80DE;&#x635F;&#x4F24;&#x7684;&#x7EC6;&#x80DE;&#x53BB;&#x6781;&#x5316;&#x81F4;&#x81EA;&#x8513;&#x5EF6;&#x6CE2;&#x3002;&#x6211;&#x4EEC;&#x4F7F;&#x7528;&#x6D3B;&#x8001;&#x9F20;&#x76AE;&#x8D28;&#x4E2D;&#x7684;&#x53CC;&#x5149;&#x5B50;&#x663E;&#x5FAE;&#x955C; NADH &#x548C;&#x6C27;&#x4F20;&#x611F;&#x5668;&#x7535;&#x6781;&#xFF0C;&#x627E;&#x5230;&#x60E9;&#x6559;&#x7F72;&#x94FE;&#x63A5;&#x5230;&#x4E25;&#x91CD;&#x7F3A;&#x6C27;&#x548C;&#x6807;&#x8BB0;&#x795E;&#x7ECF;&#x5143;&#x80BF;&#x80C0;&#xFF0C;&#x53EF;&#x957F;&#x8FBE;&#x51E0;&#x5206;&#x949F;&#x7684;&#x65F6;&#x95F4;&#x3002;&#x6811;&#x7A81;&#x72B6;&#x7ED3;&#x6784;&#x548C;&#x523A;&#x5728;&#x60E9;&#x6559;&#x7F72;&#x671F;&#x95F4;&#x635F;&#x5931;&#x7684;&#x53D8;&#x5316;&#x5E76;&#x4E0D;&#x900A;&#x8272;&#x4E8E;&#x5728;&#x7F3A;&#x6C27;&#x7684;&#x53BB;&#x6781;&#x5316;&#x8FC7;&#x7A0B;&#x3002;&#x63D0;&#x9AD8; O2 &#x53EF;&#x7528;&#x6027;&#x60E9;&#x6559;&#x7F72;&#x7684;&#x6301;&#x7EED;&#x65F6;&#x95F4;&#x7F29;&#x77ED;&#x4E86;&#xFF0C;&#x63D0;&#x9AD8;&#x4E86;&#x5F53;&#x5730;&#x7684;&#x6C27;&#x5316;&#x8FD8;&#x539F;&#x72B6;&#x6001;&#x3002;&#x6211;&#x4EEC;&#x7684;&#x7814;&#x7A76;&#x7ED3;&#x679C;&#x8868;&#x660E;&#x5173;&#x8054;&#x60E9;&#x6559;&#x7F72;&#x7684;&#x7EC4;&#x7EC7;&#x7F3A;&#x6C27;&#x9020;&#x6210;&#x7684;&#x77AC;&#x6001; O2 &#x9700;&#x6C42;&#x8D85;&#x8FC7;&#x8840;&#x7BA1; O2 &#x4F9B;&#x5E94;&#x589E;&#x52A0;&#x3002;

&#x76AE;&#x5C42;&#x548C;&#x539F;&#x56E0;&#x4E0E;&#x7EC4;&#x7EC7;&#x7F3A;&#x6C27;&#x4E0D;&#x8C0B;&#x800C;&#x5408;&#x3002;

D&#xE9;pression corticale propagation (CDD) est une onde qui s&#x26;#39;auto-propagent de la d&#xE9;polarisation cellulaire qui a &#xE9;t&#xE9; impliqu&#xE9; dans la migraine et une l&#xE9;sion neuronale progressive apr&#xE8;s un AVC et les traumatismes cr&#xE2;niens. Utilisation de l&#x26;#39;imagerie &#xE0; deux photons NADH microscopique et micro&#xE9;lectrodes capteur d&#x26;#39;oxyg&#xE8;ne dans le cortex de la souris en direct, nous constatons que la CDD est li&#xE9;e &#xE0; une hypoxie s&#xE9;v&#xE8;re et un gonflement marqu&#xE9; neuronale qui peut durer jusqu&#x26;#39;&#xE0; plusieurs minutes. Changements dans les structures dendritiques et la perte des &#xE9;pines durant la CDD sont comparables &#xE0; celles pendant la d&#xE9;polarisation anoxique. Accro&#xEE;tre la disponibilit&#xE9; O2 raccourcit la dur&#xE9;e de la CDD et am&#xE9;liore l&#x26;#39;&#xE9;tat d&#x26;#39;oxydor&#xE9;duction locale. Nos r&#xE9;sultats indiquent que l&#x26;#39;hypoxie tissulaire associ&#xE9;e &#xE0; la CDD est caus&#xE9;e par une augmentation transitoire de la demande en O2 sup&#xE9;rieure &#xE0; l&#x26;#39;offre O2 vasculaire.

D&#xE9;pression corticale &#xE9;pandage Causes et co&#xEF;ncide avec l&#x26;#39;hypoxie tissulaire

Kortikale spreading Depression (CSD) ist ein b&ouml;sartiger Welle der zellul&auml;ren Depolarisation, die Migr&auml;ne und progressive neuronale Sch&auml;digung nach Schlaganfall und Kopf-Trauma hat verwickelt war. Mit zwei-Photonen mikroskopische NADH Bildgebung und Sauerstoff-Sensor Mikroelektroden im Leben Maus Kortex, finden wir, dass CSD mit schweren Hypoxie verbunden ist und markierte neuronale Schwellung, die kann bis zu mehreren Minuten dauern. Ver&auml;nderungen der dendritischen Strukturen und Verlust von Dornen w&auml;hrend der CSD sind vergleichbar mit denen bei anoxischen Depolarisation. Die zunehmende Verf&uuml;gbarkeit von O2 verk&uuml;rzt die Dauer des CSD und verbessert die lokale Redox-Zustand. Unsere Ergebnisse zeigen, dass diese Gewebehypoxie zugeordnete CSD durch einen vor&uuml;bergehenden Anstieg der O2-Bedarf von mehr als vaskul&auml;re O2-Versorgung verursacht wird.

Kortikale spreading Depression verursacht und deckt sich mit Gewebehypoxie.

Depressione di diffusione corticale (CSD) &egrave; un&#39;onda di depolarizzazione cellulare che &egrave; stato implicato nell&#39;emicrania e nella progressiva lesione neuronale dopo trauma stroke e testa auto-moltiplicazione. Utilizzando due-fotone microscopica NADH imaging e microelettrodi sensore ossigeno nella corteccia del mouse dal vivo, troviamo che CSD &egrave; legata a grave ipossia e marcato gonfiore neuronale che pu&ograve; durare fino a diversi minuti. Cambiamenti nelle strutture dendritiche e perdita delle spine durante CSD sono paragonabili a quelli durante la depolarizzazione anossica. Aumentando la disponibilit&agrave; di O2 si riduce la durata del CSD e migliora stato redox locale. I nostri risultati indicano che ipossia tissutale associato CSD &egrave; causata da un transitorio aumento della domanda di O2 superiore al rifornimento di O2 vascolare.

Depressione di diffusione corticale provoca e coincide con ipossia tissutale.

&#x76AE;&#x8CEA;&#x62E1;&#x6563;&#x3046;&#x3064;&#x75C5;&#xFF08;CSD&#xFF09;&#x306F;&#x3001;&#x8133;&#x5352;&#x4E2D;&#x3084;&#x982D;&#x90E8;&#x5916;&#x50B7;&#x5F8C;&#x306E;&#x982D;&#x75DB;&#x3067;&#x3001;&#x30D7;&#x30ED;&#x30B0;&#x30EC;&#x30C3;&#x30B7;&#x30D6;&#x795E;&#x7D4C;&#x7D30;&#x80DE;&#x50B7;&#x5BB3;&#x306B;&#x95A2;&#x4E0E;&#x3057;&#x3066;&#x3044;&#x308B;&#x7D30;&#x80DE;&#x306E;&#x8131;&#x5206;&#x6975;&#x306E;&#x81EA;&#x5DF1;&#x4F1D;&#x64AD;&#x6CE2;&#x3067;&#x3042;&#x308B;&#x3002;&#x30E9;&#x30A4;&#x30D6;&#x306E;&#x30DE;&#x30A6;&#x30B9;&#x76AE;&#x8CEA;&#x3067;&#x306E;2&#x5149;&#x5B50;&#x9855;&#x5FAE;&#x93E1;NADH&#x30A4;&#x30E1;&#x30FC;&#x30B8;&#x30F3;&#x30B0;&#x3068;&#x9178;&#x7D20;&#x30BB;&#x30F3;&#x30B5;&#x306E;&#x96FB;&#x6975;&#x3092;&#x4F7F;&#x7528;&#x3057;&#x3066;&#x3001;&#x6211;&#x3005;&#x306F;&#x3001;CSD&#x304C;&#x6DF1;&#x523B;&#x306A;&#x4F4E;&#x9178;&#x7D20;&#x72B6;&#x614B;&#x3068;&#x3044;&#x304F;&#x3064;&#x304B;&#x306E;&#x5206;&#x307E;&#x3067;&#x7D9A;&#x304F;&#x3053;&#x3068;&#x304C;&#x3067;&#x304D;&#x308B;&#x9855;&#x8457;&#x306A;&#x795E;&#x7D4C;&#x7D30;&#x80DE;&#x306E;&#x81A8;&#x6F64;&#x306B;&#x30EA;&#x30F3;&#x30AF;&#x3055;&#x308C;&#x3066;&#x3044;&#x308B;&#x3053;&#x3068;&#x304C;&#x308F;&#x304B;&#x308A;&#x307E;&#x3059;&#x3002;&#x6A39;&#x72B6;&#x7A81;&#x8D77;&#x69CB;&#x9020;&#x306E;&#x5909;&#x5316;&#x3068;CSD&#x306E;&#x9593;&#x306B;&#x68D8;&#x306E;&#x640D;&#x5931;&#x306F;&#x3001;&#x7121;&#x9178;&#x7D20;&#x8131;&#x5206;&#x6975;&#x6642;&#x306E;&#x3082;&#x306E;&#x3068;&#x540C;&#x7B49;&#x3067;&#x3042;&#x308B;&#x3002; O2&#x306E;&#x53EF;&#x7528;&#x6027;&#x3092;&#x5897;&#x52A0;&#x3055;&#x305B;&#x308B;&#x3068;&#x3001;CSD&#x306E;&#x671F;&#x9593;&#x3092;&#x77ED;&#x7E2E;&#x3057;&#x3001;&#x5730;&#x5143;&#x306E;&#x30EC;&#x30C9;&#x30C3;&#x30AF;&#x30B9;&#x72B6;&#x614B;&#x3092;&#x5411;&#x4E0A;&#x3055;&#x305B;&#x307E;&#x3059;&#x3002;&#x6211;&#x3005;&#x306E;&#x7D50;&#x679C;&#x306F;&#x3001;CSD&#x306B;&#x95A2;&#x9023;&#x4ED8;&#x3051;&#x3089;&#x308C;&#x305F;&#x7D44;&#x7E54;&#x306E;&#x4F4E;&#x9178;&#x7D20;&#x75C7;&#x306F;&#x3001;&#x8840;&#x7BA1;O2&#x4F9B;&#x7D66;&#x91CF;&#x3092;&#x8D85;&#x3048;O2&#x9700;&#x8981;&#x306E;&#x4E00;&#x6642;&#x7684;&#x306A;&#x5897;&#x52A0;&#x306B;&#x3088;&#x3063;&#x3066;&#x5F15;&#x304D;&#x8D77;&#x3053;&#x3055;&#x308C;&#x308B;&#x3053;&#x3068;&#x3092;&#x793A;&#x3057;&#x3066;&#x3044;&#x307E;&#x3059;&#x3002;

&#x76AE;&#x8CEA;&#x62E1;&#x6563;&#x3046;&#x3064;&#x75C5;&#x306F;&#x539F;&#x56E0;&#x3068;&#x7D44;&#x7E54;&#x4F4E;&#x9178;&#x7D20;&#x3068;&#x4E00;&#x81F4;

&#xB300;&#xB1CC; &#xD53C; &#xC9C8;&#xC758; &#xC6B0;&#xC6B8;&#xC99D; &#xD655;&#xC0B0; (CSD)&#xB294; &#xD3B8;&#xB450;&#xD1B5;&#xACFC; &#xB1CC;&#xC878;&#xC911;&#xACFC; &#xBA38;&#xB9AC; &#xC678;&#xC0C1; &#xD6C4; &#xC9C4;&#xBCF4;&#xC801;&#xC778; &#xC2E0;&#xACBD; &#xC0C1;&#xD574; &#xC5F0;&#xB8E8; &#xB418;&#xC5B4; &#xC138;&#xD3EC;&#xC9C8; depolarization&#xC758; &#xC790;&#xAC00; &#xBB3C;&#xACB0;. &#xC6B0;&#xB9AC;&#xAC00; &#xC2EC;&#xAC01;&#xD55C; hypoxia CSD &#xC5F0;&#xACB0; &#xD558; &#xACE0; &#xBA87; &#xBD84; &#xC9C0;&#xB09C; &#xC218; &#xC788;&#xC2B5;&#xB2C8;&#xB2E4; &#xD45C;&#xC2DC; &#xB41C; &#xC2E0;&#xACBD; &#xBD93;&#xAE30;&#xB97C; &#xCC3E;&#xC744; &#xB450; &#xAD11;&#xC790; &#xD604;&#xBBF8;&#xACBD; NADH &#xC774;&#xBBF8;&#xC9D5; &#xBC0F; &#xC0B0;&#xC18C; &#xC13C;&#xC11C; microelectrodes&#xB97C; &#xC0AC;&#xC6A9; &#xD558; &#xC5EC; &#xB77C;&#xC774;&#xBE0C; &#xB9C8;&#xC6B0;&#xC2A4; &#xD53C; &#xC9C8;&#xC5D0;&#xC11C;. &#xC218;&#xC9C0;&#xC0C1; &#xAD6C;&#xC870;&#xC640; CSD &#xB3D9;&#xC548; &#xB4F1;&#xBF08;&#xC758; &#xC190;&#xC2E4; &#xBCC0;&#xD654; &#xBB34;&#xC0B0; &#xC18C; depolarization &#xB3D9;&#xC548; &#xC0AC;&#xB78C;&#xB4E4;&#xC5D0; &#xAC8C; &#xBE44;&#xAD50;&#xD560; &#xC218; &#xC788;&#xC2B5;&#xB2C8;&#xB2E4;. O 2&#xC758; &#xAC00;&#xC6A9;&#xC131;&#xC744; &#xB192;&#xC774;&#xAE30; CSD&#xC758; &#xAE30;&#xAC04;&#xC744; &#xB2E8;&#xCD95; &#xD558; &#xACE0; &#xC9C0;&#xBC29; &#xC0B0;&#xD654; &#xC0C1;&#xD0DC;&#xB97C; &#xD5A5;&#xC0C1; &#xC2DC;&#xD0B5;&#xB2C8;&#xB2E4;. &#xC6B0;&#xB9AC;&#xC758; &#xACB0;&#xACFC; CSD&#xC640; &#xAD00;&#xB828; &#xB41C; &#xC870;&#xC9C1;&#xC758; &#xC0B0;&#xC18C;&#xB294; &#xD608;&#xAD00; o 2 &#xACF5;&#xAE09;&#xC744; &#xCD08;&#xACFC; &#xD558;&#xB294; o 2 &#xC218;&#xC694;&#xC758; &#xC77C;&#xC2DC;&#xC801; &#xC99D;&#xAC00; &#xC758;&#xD574; &#xBC1C;&#xC0DD;&#xC744; &#xB098;&#xD0C0;&#xB0C5;&#xB2C8;&#xB2E4;.

&#xB300;&#xB1CC; &#xD53C; &#xC9C8;&#xC758; &#xD655;&#xC0B0; &#xC6B0;&#xC6B8;&#xC99D; &#xC6D0;&#xC778;&#xACFC; &#xC870;&#xC9C1; &#xC800; &#xC0B0;&#xC18C; &#xC99D;&#xACFC; &#xC77C;&#xCE58; &#xD55C;&#xB2E4;.

La depresi&#xF3;n cortical propagada (CSD) es una ola de auto-propagaci&#xF3;n de la despolarizaci&#xF3;n celular que ha sido implicado en la migra&#xF1;a y en el da&#xF1;o neuronal progresiva despu&#xE9;s del accidente cerebrovascular y el traumatismo craneoencef&#xE1;lico. Uso de dos fotones de im&#xE1;genes microsc&#xF3;picas y NADH microelectrodos del sensor de ox&#xED;geno en la corteza del rat&#xF3;n vivo, nos encontramos con que la CDS est&#xE1; vinculada a la hipoxia severa e inflamaci&#xF3;n marcada neuronal que puede durar hasta varios minutos. Cambios en las estructuras dendr&#xED;ticas y la p&#xE9;rdida de espinas en la CDS son comparables a los durante la despolarizaci&#xF3;n an&#xF3;xica. Aumento de la disponibilidad de O2 acorta la duraci&#xF3;n de la CDS y mejora el estado redox local. Nuestros resultados indican que la hipoxia tisular asociado a la CSD es causado por un aumento transitorio de la demanda de O2 superior vascular suministro de O2.

Difusi&#xF3;n de la depresi&#xF3;n cortical Causas y coincide con la hipoxia tisular

Spinal cord injury (SCI) is often complicated by secondary injury as a result of the innate inflammatory response to tissue trauma and swelling. Previous studies have shown that excessive ATP release from peritraumatic regions contributes to the inflammatory response to SCI by activation of low-affinity P2X7 receptors. Because connexin hemichannels constitute an important route for astrocytic ATP release, we here evaluated the impact on post-traumatic ATP release of deletion of connexins (Cx30/Cx43) in astrocytes. In vivo bioluminescence imaging showed a significant reduction in ATP release after weight-drop injury in mice with deletion of Cx43 compared with Cx43-expressing littermates, both on a Cx30 knockout background. Moreover, astrogliosis and microglia activation were reduced in peritraumatic areas of those mice lacking Cx43; motor recovery was also significantly improved, and the traumatic lesion was smaller. Combined, these observations are consistent with a contribution by astrocytic hemichannels to post-traumatic ATP release that aggravates secondary injury and restrains functional recovery after experimental spinal cord injury. Connexins may thereby constitute a new therapeutic target in spinal cord injury.

Critical role of connexin 43 in secondary expansion of traumatic spinal cord injury.

Calcium signaling represents the principle pathway by which astrocytes respond to neuronal activity. General anesthetics are routinely used in clinical practice to induce a sleep-like state, allowing otherwise painful procedures to be performed. Anesthetic drugs are thought to mainly target neurons in the brain and act by suppressing synaptic activity. However, the direct effect of general anesthesia on astrocyte signaling in awake animals has not previously been addressed. This is a critical issue, because calcium signaling may represent an essential mechanism through which astrocytes can modulate synaptic activity. In our study, we performed calcium imaging in awake head-restrained mice and found that three commonly used anesthetic combinations (ketamine/xylazine, isoflurane, and urethane) markedly suppressed calcium transients in neocortical astrocytes. Additionally, all three anesthetics masked potentially important features of the astrocyte calcium signals, such as synchronized widespread transients that appeared to be associated with arousal in awake animals. Notably, anesthesia affected calcium transients in both processes and soma and depressed spontaneous signals, as well as calcium responses, evoked by whisker stimulation or agonist application. We show that these calcium transients are inositol 1,4,5-triphosphate type 2 receptor (IP(3)R2)-dependent but resistant to a local blockade of glutamatergic or purinergic signaling. Finally, we found that doses of anesthesia insufficient to affect neuronal responses to whisker stimulation selectively suppressed astrocyte calcium signals. Taken together, these data suggest that general anesthesia may suppress astrocyte calcium signals independently of neuronal activity. We propose that these glial effects may constitute a nonneuronal mechanism for sedative action of anesthetic drugs.

General anesthesia selectively disrupts astrocyte calcium signaling in the awake mouse cortex.

The nonspecific and variable presentation of traumatic brain injury (TBI) has motivated an intense search for blood-based biomarkers that can objectively predict the severity of injury. However, it is not known how cytosolic proteins released from traumatized brain tissue reach the peripheral blood. Here we show in a murine TBI model that CSF movement through the recently characterized glymphatic pathway transports biomarkers to blood via the cervical lymphatics. Clinically relevant manipulation of glymphatic activity, including sleep deprivation and cisternotomy, suppressed or eliminated TBI-induced increases in serum S100β, GFAP, and neuron specific enolase. We conclude that routine TBI patient management may limit the clinical utility of blood-based biomarkers because their brain-to-blood transport depends on glymphatic activity. 

Biomarkers of traumatic injury are transported from brain to blood via the glymphatic system.

Energy production in the brain depends almost exclusively on oxidative metabolism. Neurons have small energy reserves and require a continuous supply of oxygen (O2). It is therefore not surprising that one of the hallmarks of normal brain function is the tight coupling between cerebral blood flow and neuronal activity. Since capillaries are embedded in the O2-consuming neuropil, we have here examined whether activity-dependent dips in O2 tension drive capillary hyperemia. In vivo analyses showed that transient dips in tissue O2 tension elicit capillary hyperemia. Ex vivo experiments revealed that red blood cells (RBCs) themselves act as O2 sensors that autonomously regulate their own deformability and thereby flow velocity through capillaries in response to physiological decreases in O2 tension. This observation has broad implications for understanding how local changes in blood flow are coupled to synaptic transmission. 

Erythrocytes Are Oxygen-Sensing Regulators of the Cerebral Microcirculation.

Astrocytes have in recent years become the focus of intense experimental interest, yet markers for their definitive identification remain both scarce and imperfect. Astrocytes may be recognized as such by their expression of glial fibrillary acidic protein, glutamine synthetase, glutamate transporter 1 (GLT1), aquaporin-4, aldehyde dehydrogenase 1 family member L1, and other proteins. However, these proteins may all be regulated both developmentally and functionally, restricting their utility. To identify a nuclear marker pathognomonic of astrocytic phenotype, we assessed differential RNA expression by FACS-purified adult astrocytes and, on that basis, evaluated the expression of the transcription factor SOX9 in both mouse and human brain. We found that SOX9 is almost exclusively expressed by astrocytes in the adult brain except for ependymal cells and in the neurogenic regions, where SOX9 is also expressed by neural progenitor cells. Transcriptome comparisons of SOX9+ cells with GLT1+ cells showed that the two populations of cells exhibit largely overlapping gene expression. Expression of SOX9 did not decrease during aging and was instead upregulated by reactive astrocytes in a number of settings, including a murine model of amyotrophic lateral sclerosis (SOD1G93A), middle cerebral artery occlusion, and multiple mini-strokes. We quantified the relative number of astrocytes using the isotropic fractionator technique in combination with SOX9 immunolabeling. The analysis showed that SOX9+ astrocytes constitute ∼10-20% of the total cell number in most CNS regions, a smaller fraction of total cell number than previously estimated in the normal adult brain. Astrocytes are traditionally identified immunohistochemically by antibodies that target cell-specific antigens in the cytosol or plasma membrane. We show here that SOX9 is an astrocyte-specific nuclear marker in all major areas of the CNS outside of the neurogenic regions. Based on SOX9 immunolabeling, we document that astrocytes constitute a smaller fraction of total cell number than previously estimated in the normal adult mouse brain.

Nanhong Lou

Center for Translational Neuromedicine,
Division of Glial Therapeutics,
Center for Translational Neuromedicine, Division of Glial Therapeutics

University of Rochester Medical Center

Cannula Implantation into the Cisterna Magna of Rodents

Nanhong Lou

Center for Translational Neuromedicine,Division of Glial Therapeutics,Center for Translational Neuromedicine, Division of Glial Therapeutics

University of Rochester Medical Center

Cannula Implantation into the Cisterna Magna of Rodents

Center for Translational Neuromedicine,
Division of Glial Therapeutics,
Center for Translational Neuromedicine, Division of Glial Therapeutics