Hypoglycemia Induces Transient Neurogenesis and Subsequent … · 2005-01-18 · Hypoglycemia...

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Hypoglycemia Induces Transient Neurogenesis and Subsequent Progenitor Cell Loss in the Rat Hippocampus Sang Won Suh, 1,2 Yang Fan, 2,3 Shwuhuey M. Hong, 2,3 Zhengyan Liu, 2,3 Yasuhiko Matsumori, 2,3 Philip R. Weinstein, 2,3 Raymond A. Swanson, 1,2 and Jialing Liu 2,3 Neurogenesis after brain injury not only leads to the replacement of damaged cells but might also contribute to functional recovery, suggesting the possibility of endogenous neural repair. We investigated the extent of hippocampal neural regeneration in a rat model of hypoglycemia. Two weeks after 30 min of insulin-in- duced isoelectric electroencephalogram, extensive neu- ronal loss was observed in the hippocampus, including area CA1 and dentate gyrus (DG). A transient increase in progenitor cell proliferation in the DG subgranular zone (SGZ) was detected, leading to an increase of immature neuroblasts 1–2 weeks after hypoglycemic insult. Most of the surviving newborn cells assumed a neuronal phenotype within 1 month in DG, a few cells near the site of granule-cell death becoming astroglia or microglia. No neuronal regeneration was observed in the CA1 after hypoglycemia, although dividing cells appeared to be astroglia or microglia in CA1 and dentate hilus. At 4 weeks after hypoglycemia, prolifer- ative activity in the SGZ diminished below baseline in experimental versus control rats, with a subsequent reduction of neuroblasts. Morphological findings (dou- blecortin staining) suggest permanent progenitor cell loss in some areas of SGZ. Reduced neurogenesis in DG and lack of neuronal regeneration in CA1 may impede cognitive recovery after severe hypoglycemia injury. Diabetes 54:500 –509, 2005 N eural stem cells have been implicated in pro- cesses that lead to neural regeneration follow- ing central nervous system diseases and injuries, raising the possibility that it might be feasible to ameliorate brain injury by boosting endogenous neural regeneration (1– 4). Neurogenesis occurs in the hippocampus of all mammals, including humans (5,6). Recent data show that newly born cells become function- ally integrated into the dentate gyrus (DG) and have passive membrane properties, action potentials, and func- tional synaptic inputs similar to those found in mature dentate granule cells (7). Most important are findings that newly generated neurons play a significant role in synaptic plasticity (8) and that a reduction in the number of these cells impairs learning and memory (9). Our recent evi- dences suggest that neurogenesis following ischemic brain injury not only leads to the replacement of damaged cells, but might also contribute to functional recovery (10,11), thereby further supporting the notion that strategies for augmenting endogenous neurogenesis may hold clues for the development of restorative therapy. Hypoglycemic brain injury is a common and serious complication of insulin therapy that may also occur during tight management of blood glucose levels with other hypoglycemic agents. Similar to cerebral ischemia, in- creased aspartate and glutamate levels during and after hypoglycemia cause a sustained activation of glutamate receptors that results in an excitotoxic neuropathology in which certain neurons are selectively killed (12). Patho- logical studies in humans and animals show that hypogly- cemia-induced neuronal death occurs preferentially in the CA1, subiculum, and DG of the hippocampus, superficial layers of the cortex, and striatum (12). Because of the extensive neuronal loss, one of the neurological sequalae associated with hypoglycemia is cognitive decline. Ac- cording to clinical studies, significant learning and mem- ory deficits correlate with the frequency of hypoglycemia not only in patients with type 1 diabetes, but also in the relatively younger group among the population with type 2 diabetes (13). The prevalence of severe hypoglycemia, including hypoglycemic coma, could be as high as 40.5% in patients with type 1 diabetes alone (14). In support of such clinical observations are studies in which rats had signif- icant deficits in spatial learning and memory with associ- ated hippocampal neuronal loss in an experimental model of hypoglycemia (15). As there is no effective treatment for hypoglycemia-caused cognitive impairment, enhancing neurogenesis might provide a novel therapeutic strategy for treating patients with hypoglycemia-induced brain damage and functional impairment. Despite a wealth of literature on the neurochemistry and neuropathology of hypoglycemia, little is known about the From the 1 Department of Neurology, University of California at San Fran- cisco, San Francisco, California; the 2 Department of Neurological Surgery, University of California at San Francisco, San Francisco; and the 3 San Francisco Department of Veterans Affairs Medical Center, San Francisco, California. Address correspondence and reprint requests to Dr. Jialing Liu, Department of Neurological Surgery (112C), University of California at San Francisco and Department of Veterans Affairs Medical Center, 4150 Clement St., San Francisco, California 94121. E-mail: [email protected]. Received for publication 12 August 2004 and accepted in revised form 1 November 2004. DCX, double cortin; DG, dentate gyrus; EEG, electroencephalogram; GCL, granule cell layer; GFAP, glial fibrillary acidic protein; SGZ, subgranular zone; SVZ, subventricular zone. © 2005 by the American Diabetes Association. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. 500 DIABETES, VOL. 54, FEBRUARY 2005

Transcript of Hypoglycemia Induces Transient Neurogenesis and Subsequent … · 2005-01-18 · Hypoglycemia...

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Hypoglycemia Induces Transient Neurogenesis andSubsequent Progenitor Cell Loss in the RatHippocampusSang Won Suh,

1,2Yang Fan,

2,3Shwuhuey M. Hong,

2,3Zhengyan Liu,

2,3Yasuhiko Matsumori,

2,3

Philip R. Weinstein,2,3

Raymond A. Swanson,1,2

and Jialing Liu2,3

Neurogenesis after brain injury not only leads to thereplacement of damaged cells but might also contributeto functional recovery, suggesting the possibility ofendogenous neural repair. We investigated the extent ofhippocampal neural regeneration in a rat model ofhypoglycemia. Two weeks after 30 min of insulin-in-duced isoelectric electroencephalogram, extensive neu-ronal loss was observed in the hippocampus, includingarea CA1 and dentate gyrus (DG). A transient increasein progenitor cell proliferation in the DG subgranularzone (SGZ) was detected, leading to an increase ofimmature neuroblasts 1–2 weeks after hypoglycemicinsult. Most of the surviving newborn cells assumed aneuronal phenotype within 1 month in DG, a few cellsnear the site of granule-cell death becoming astroglia ormicroglia. No neuronal regeneration was observed inthe CA1 after hypoglycemia, although dividing cellsappeared to be astroglia or microglia in CA1 anddentate hilus. At 4 weeks after hypoglycemia, prolifer-ative activity in the SGZ diminished below baseline inexperimental versus control rats, with a subsequentreduction of neuroblasts. Morphological findings (dou-blecortin staining) suggest permanent progenitor cellloss in some areas of SGZ. Reduced neurogenesis in DGand lack of neuronal regeneration in CA1 may impedecognitive recovery after severe hypoglycemia injury.Diabetes 54:500–509, 2005

Neural stem cells have been implicated in pro-cesses that lead to neural regeneration follow-ing central nervous system diseases andinjuries, raising the possibility that it might be

feasible to ameliorate brain injury by boosting endogenous

neural regeneration (1–4). Neurogenesis occurs in thehippocampus of all mammals, including humans (5,6).Recent data show that newly born cells become function-ally integrated into the dentate gyrus (DG) and havepassive membrane properties, action potentials, and func-tional synaptic inputs similar to those found in maturedentate granule cells (7). Most important are findings thatnewly generated neurons play a significant role in synapticplasticity (8) and that a reduction in the number of thesecells impairs learning and memory (9). Our recent evi-dences suggest that neurogenesis following ischemic braininjury not only leads to the replacement of damaged cells,but might also contribute to functional recovery (10,11),thereby further supporting the notion that strategies foraugmenting endogenous neurogenesis may hold clues forthe development of restorative therapy.

Hypoglycemic brain injury is a common and seriouscomplication of insulin therapy that may also occur duringtight management of blood glucose levels with otherhypoglycemic agents. Similar to cerebral ischemia, in-creased aspartate and glutamate levels during and afterhypoglycemia cause a sustained activation of glutamatereceptors that results in an excitotoxic neuropathology inwhich certain neurons are selectively killed (12). Patho-logical studies in humans and animals show that hypogly-cemia-induced neuronal death occurs preferentially in theCA1, subiculum, and DG of the hippocampus, superficiallayers of the cortex, and striatum (12). Because of theextensive neuronal loss, one of the neurological sequalaeassociated with hypoglycemia is cognitive decline. Ac-cording to clinical studies, significant learning and mem-ory deficits correlate with the frequency of hypoglycemianot only in patients with type 1 diabetes, but also in therelatively younger group among the population with type 2diabetes (13). The prevalence of severe hypoglycemia,including hypoglycemic coma, could be as high as 40.5% inpatients with type 1 diabetes alone (14). In support of suchclinical observations are studies in which rats had signif-icant deficits in spatial learning and memory with associ-ated hippocampal neuronal loss in an experimental modelof hypoglycemia (15). As there is no effective treatment forhypoglycemia-caused cognitive impairment, enhancingneurogenesis might provide a novel therapeutic strategyfor treating patients with hypoglycemia-induced braindamage and functional impairment.

Despite a wealth of literature on the neurochemistry andneuropathology of hypoglycemia, little is known about the

From the 1Department of Neurology, University of California at San Fran-cisco, San Francisco, California; the 2Department of Neurological Surgery,University of California at San Francisco, San Francisco; and the 3SanFrancisco Department of Veterans Affairs Medical Center, San Francisco,California.

Address correspondence and reprint requests to Dr. Jialing Liu, Departmentof Neurological Surgery (112C), University of California at San Francisco andDepartment of Veterans Affairs Medical Center, 4150 Clement St., SanFrancisco, California 94121. E-mail: [email protected].

Received for publication 12 August 2004 and accepted in revised form 1November 2004.

DCX, double cortin; DG, dentate gyrus; EEG, electroencephalogram; GCL,granule cell layer; GFAP, glial fibrillary acidic protein; SGZ, subgranular zone;SVZ, subventricular zone.

© 2005 by the American Diabetes Association.The costs of publication of this article were defrayed in part by the payment of page

charges. This article must therefore be hereby marked “advertisement” in accordance

with 18 U.S.C. Section 1734 solely to indicate this fact.

500 DIABETES, VOL. 54, FEBRUARY 2005

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potential for neurological repair and regeneration follow-ing hypoglycemic brain damage. A recent experimentalmodel of repetitive hypoglycemia with mild damage to theparasagital cortex failed to show neurogenesis in the DG(16), suggesting that the extent and location of brain injurymight determine whether regeneration occurs. Granulecell death, seen in both global ischemia and epilepsy, isassociated with increased progenitor cell proliferation inthe subgranular zone (SGZ), further supporting our hy-pothesis that hippocampal cell loss might be required toinduce neurogenesis in the DG. Here we investigatewhether progenitor cell proliferation and neurogenesisoccur in the hippocampus in a rat model of hypoglycemia.Our results demonstrate a transient increase in progenitorcell proliferation in the SGZ and neurogenesis in thegranule cell layer (GCL) during the first 2 weeks afterhypoglycemia, with subsequent loss of progenitor cells inthe SGZ beginning 4 weeks after the insult. Unlike findingsreported in regard to cerebral global ischemia (17), no

neuronal regeneration was detected in the CA1 properafter hypoglycemia. Apart from extensive cell loss in manyhippocampal regions, including the CA1, reduced neuronalreplacement due to progenitor cell loss in the DG mightlimit cognitive recovery after hypoglycemic brain injury.

RESEARCH DESIGNS AND METHODS

Animal surgery and hypoglycemia induction. Two-month-old maleSprague-Dawley rats (Charles River, CA) were housed and cared for accord-ing to federal and institutional guidelines. Hypoglycemia induction wasperformed as described (15); sham-treated control rats underwent all proce-dures except insulin injection. Rats were kept under anesthesia following theinduction of hypoglycemia until 3 h after the onset of glucose infusion topermit monitoring and control of physiological parameters. A femoral arteryline was placed for blood pressure monitoring (BIOPAC System, SantaBarbara, CA) and blood sampling. Blood gas was measured at 1-h intervals byI-STAT (Princeton, NJ). Tidal volume and oxygen flow were adjusted via asmall animal respirator (Harvard Apparatus, South Natick, MA) to keep PaCO2

between 35 and 45 mmHg and PaO2 �100 mmHg. For electroencephalogram(EEG) monitoring (BIOPAC), burr holes were made in the skull bilaterallyover parietal cortex and monopolar electrodes placed beneath the dura. A

FIG. 1. Hypoglycemia induces EEG, heart rate, and bloodpressure changes and subsequent widespread neuronalloss. A: Hypoglycemia induces isoelectric EEG and in-creased blood pressure and heart rate. a., Normal restingEEG pattern. b., EEG pattern slowed down with increasedamplitude after the onset of hypoglycemia. Blood pressureand heart rate were still within the normal range. c., EEGshowed isoelectric pattern. Mean arterial blood pressureand heart rate increased up to 180 mmHg and 400/min,respectively. d., EEG, blood pressure, and heart rate com-pletely normalized 60 min after glucose infusion. B: Hypo-glycemia (HG)-induced wide spread neuronal loss wasdetected by NeuN immunohistochemistry at 2 weeks fol-lowing 30 min of isoelectric EEG and sham treatment.Representative photomicrographs detected apparent neu-ronal loss (indicated by arrowheads) in the subiculum,CA1 pyramidal layer, tip and upper blade of the DG,striatum (ST), and superficial layers of the cerebral cor-tex (CTX). Scale bars, 250 �m.

TABLE 1Stereological estimate of the total number of neurons, volume, and cell density in the septal CA1 of adult male rats in sham-operatedcontrol and hypoglycemia groups

Sham HG-2W HG-4W

Cell number (�104) 20.5 � 0.09 5.19 � 1.52* 5.95 � 1.27*Volume (mm3) 0.66 � 0.03 0.27 � 0.03† 0.39 � 0.02†Cell density (�105 cells/mm3) 3.14 � 0.03 1.76 � 0.26† 1.48 � 0.24†

Data are means � SD per hemisphere. n � 4 in sham-operated control group (Sham) and n � 7 in each hypoglycemia group (HG). An opticalfractionator was used to estimate the total numbers of NeuN immunoreactive cells in the septal CA1. Significant difference in cell number,volume, and density was found at 2 weeks (HG-2W) and 4 weeks (HG-4W) after hypoglycemia when compared with controls, as indicated.*P � 0.001, †P � 0.005.

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reference needle was placed in neck muscle. A heating blanket/rectal probeservo-loop was used to maintain core temperature at 36.5–37.5°C until the ratshad recovered from anesthesia, at which time they were euthermic. Hypogly-cemia was induced by intraperitoneal injection of 15 units/kg insulin (Novo-lin-R; Novo Nordisk). Blood glucose was measured (YSI 2700 glucoseanalyzer; YSI, Yellow Springs, OH) at 30-min intervals during the induction ofhypoglycemia and at 60-min intervals during the recovery from hypoglycemia.Mean arterial blood pressure was maintained between 160 and 200 mmHgduring the entire isoelectric period by adjusting isoflurane concentration, andbradycardia during hypoglycemia was prevented with atropine (1 mg/kg;American Pharmaceutical Partners, Los Angeles, CA). Hypoglycemia wasterminated by intravenous injection of 0.2 ml of 50% glucose via the femoralvein, followed by continuous infusion of 1:1 solution of 50% glucose andKrebs-Henseleit buffer (1.5 ml/h for 3 h). No seizure activity was observed in�200 rats subjected to 30 min of isoelectric EEG.Cell proliferation and survival and phenotype determination. Ki67immunohistochemistry was used to determine the amount of cell proliferationin the SGZ at 4 days and at 1, 2, and 4 weeks after the induction ofhypoglycemia. To quantify hypoglycemia-induced cell proliferation at thesubventricular zone (SVZ), a separate group of rats received three intraperi-toneal injections of BrdU (50 mg/kg; Sigma, St. Louis, MO) 6 h apart at 4 daysafter the induction of hypoglycemia and were killed 24 h later. To investigatethe phenotype and survival of newborn cells at the GCL, rats receivedintraperitoneal injections three times daily of BrdU for 4 consecutive daysfrom 3 to 6 days following hypoglycemia and were killed 2 and 4 weeks later.

Immunohistochemistry and immunofluorescence staining. Rats wereanesthetized and transcardially perfused with 4% paraformaldehyde as de-scribed (10). Free floating coronal sections (40-�m) were immunostained asdescribed (10) using the following reagents: mouse anti-BrdU (0.25 �g/ml;Roche, Indianapolis, IN), rat anti-BrdU (2 �g/ml; Accurate Chemicals, West-bury, NY), rabbit anti-calbindin-D28k (0.1 �g/ml; SWANT, Bellinzona, Switzer-land), rabbit anti-Ki67 (recognizing nuclear antigen expressed during allproliferative stages of the cell cycle except G0 [18], 0.1 �g/ml; Novocastra,Newcastle upon Tyne, U.K.), mouse anti-NeuN (1 �g/ml; Chemicon, Temecula,CA), goat anti–double cortin (DCX) (recognizing neuroblasts [19], 0.8 �g/ml;Santa Cruz Biotechnology, Santa Cruz, CA), mouse anti–Tuj-1 (1 �g/ml;BABCO, Berkeley, CA), mouse anti-CD11b (1 �g/ml; Serotec, Raleigh, NC) ormouse anti–glial fibrillary acidic protein (GFAP) (1 �g/ml; Chemicon), biotin-ylated sheep anti-mouse and anti-rat secondary antibodies (5 �g/ml; Amer-sham, Cleveland, OH), ABC solution (Vector laboratories, Burlingame, CA),and Streptavidine Alexa Fluor 488 and Alexa Fluor 594 goat anti-mouse oranti-rabbit IgG conjugates (5 �g/ml; Molecular Probes, Eugene, OR).Confocal microscopy. Fluorescence signals were detected using the ZeissLSM 510 confocal imaging system (Zeiss, Thornwood, NY) with a sequentialscanning mode for Alexa 488 and 594. Stacks of images (1,024 � 1,024 pixels)from consecutive slices of 0.66–0.7 �m in thickness were obtained byaveraging four scans per slice and processed with Adobe Photoshop (AdobeSystems, Mountain View, CA).Cell counting. The number of NeuN-stained CA1 neurons was determined inevery 12th coronal section spanning the septal hippocampus (Bregma level

FIG. 2. Hypoglycemia induces progenitor cell proliferationand increases the amount of immature neurons in the SGZ.The numbers of proliferating cells (A and B) and immatureneurons (C) transiently increased in the SGZ during the firstweek after 30 min of isoelectric EEG. A: The number of Ki67immunoreactive cells were estimated by unbiased stereologyper hemisphere spanning the septal hippocampus of ratskilled at the time indicated. One-way ANOVA was performedon the log-transformed Ki67 counts. Significant differenceswere detected between sham-operated controls and hypogly-cemic animals at 4 days and at 1 and 4 weeks after theinduction of hypoglycemia. **P < 0.01. Representative pho-tomicrographs showing Ki67 (B) and DCX (C) immunostain-ing of proliferating cells and immature neuroblasts 1 (HG-1)and 4 (HG-4) weeks after sham and hypoglycemia (HG)treatment. At 4 weeks after hypoglycemia, diminished imma-ture neuroblasts are likely due to loss of progenitor cells(arrows). Scale bar, 250 �m.

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�1.8 to �3.8) using unbiased stereology (20) (Stereo Investigator; Micro-BrightField, Williston, VT). Counting frames (15 � 15 � 20 �m) were placedat the intersection of a matrix (30 � 30 �m for Ki67 and 200 � 200 �m forNeuN and BrdU estimation) randomly superimposed onto the region ofinterest by the program. Ki67 and BrdU immunoreactive cells were counted inthe SGZ and GCL, respectively, in the same fashion. The volume of the dentategranule layer was determined using Cavalieri’s principal (21) with a 60 � 60�m grid matrix. For quantification of BrdU immunostaining at the SVZ, themean area of BrdU immunoreactivity was determined by tracing the regions ofinterest according to the method of Parent et al. (22).Statistical analysis. Data were expressed as the mean � SE. Data wereevaluated by one-way ANOVA, followed by Tukey-Kramer post hoc tests whenappropriate. P values �0.05 were considered significant.

RESULTS

Hypoglycemia induces isoelectric EEG and wide-

spread neuronal loss. A rat model of hypoglycemia wasproduced as described (15). EEG was used to monitorbrain activity and to generate a reproducible injury pat-tern. The EEG patterns passed through progressive stagecharacteristics of severe hypoglycemia (Fig. 1A). Theisoelectric EEG pattern generally developed after bloodglucose concentrations had fallen to �0.5 mmol/l forseveral minutes, simulating hypoglycemic coma. A concur-rent increase in blood pressure and heart rate also oc-curred during isoelectric EEG. EEG, blood pressure, andheart rate recovery began 12–15 min after the onset of

glucose infusion and completely returned to baseline level60 min after glucose infusion.

Two weeks after rats underwent 30-min insulin-inducedisoelectric EEG, neuronal loss was seen in the cerebralcortex, striatum, subiculum, CA1, and DG (Fig. 1B). Thereis no apparent progressive cell loss in the CA1 during theperiod of 2–4 weeks after the insult, as indicated by thecell number and volume in this region (Table 1). Unbiasedstereology estimated a near 30% reduction in the volume ofthe DG at 4 weeks after hypoglycemia brain injury (sham3.76 � 109 � 1.56 � 108 �m3, n � 6, and hypoglycemia2.61 � 109 � 1.84 � 108 �m3, n � 6). A more extensive cellloss of 70–75% occurred in the CA1 (Table 1).Hypoglycemia induces a transient increase in progen-

itor cell proliferation and permanent progenitor cell

loss in the DG. To investigate how hypoglycemia-inducedneuronal loss in the DG affected the SGZ progenitor cellproliferation and neurogenesis, rats were injected withthymidine analog BrdU as described and were killed at 4days and at 1, 2, and 4 weeks after the induction ofhypoglycemia. Perfusion-fixed coronal rat brain sliceswere assessed by Ki67, DCX, and BrdU immunohistochem-istry. Increased cell proliferation occurred in the SGZ ofrats as early as 4 days after 30-min insulin-induced isoelec-

FIG. 3. Hypoglycemia increases the number of BrdU-labeled cells in the SGZ at 2 and 4 weeks following 30 min of isoelectric EEG. Unbiasedstereology was used to estimate BrdU immunoreactive cell number (A) and cell density (B) in the GCL 2 (2W) (n � 8 each in sham [f] andhypoglycemia [�]) and 4 (4W) (n � 8 each in sham and hypoglycemia) weeks following hypoglycemia. ****P < 0.001. There was also a decline inboth BrdU-labeled cell number and cell density between 2 and 4 weeks after hypoglycemia. **P < 0.01, ***P < 0.005. C: Representativephotomicrographs showing an increased number of labeled BrdU nuclei having migrated into the GCL at 2 and 4 weeks after hypoglycemia (HG)compared with sham controls. Fewer BrdU-labeled cells remained at 4 weeks compared with those at 2 weeks after hypoglycemia. Scale bar, 250�m.

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tric EEG, compared with sham-operated rats (Fig. 2A andB). At 2 weeks after hypoglycemia was induced, prolifer-ation in the SGZ returned to the baseline level (Fig. 2A).DCX immunostaining showed an increased number ofneuroblasts 1–2 weeks after hypoglycemia as comparedwith that in sham-operated controls (Fig. 2C).

However, Ki67 immunoreactivity detected significantlyreduced progenitor cell proliferation in the SGZ (P � 0.01)at 4 weeks after the induction of hypoglycemia (5,994 � 595)as compared with that in the sham-operated control rats(2,948 � 489) (Fig. 2A). A reduction of immature neuroblastsalso took place at 4 weeks after the induction of hypoglyce-mia (Fig. 2C). DCX immunostaining showed aberrant stain-ing patterns near the site of DG cell loss (Fig. 2C).Newborn cells survived at least 4 weeks after hypo-

glycemia. To determine how newborn cells survive in theDG and whether they integrate into the GCL, we estimatedthe number and density of BrdU-labeled cells in the GCLby unbiased stereology at 2 and 4 weeks after the induc-tion of hypoglycemia. There were significantly more BrdU-labeled cells in the GCL at both time points (Fig. 3A),corresponding to a higher density of BrdU labeling (Fig.3B), in the rats subjected to hypoglycemia than in thecontrols. Similar to findings observed after cerebral globalischemia (10), the number and density of BrdU-labeledcells at 4 weeks after hypoglycemia were also less than atthe 2-week time point, suggesting that many cells newlyborn to the DG died over time after brain injury (Fig. 3C).Most of the newborn cells differentiate into neuronal

phenotypes in the GCL. To investigate the fate ofnewborn cells in the DG after hypoglycemic insult, doubleimmunofluorecence staining with BrdU and neuronal/glialmarkers, including Tuj1, NeuN, Calbindin28kd, and GFAP,were performed, and signals were detected by confocalmicroscopy (Fig. 4). At 2 weeks after hypoglycemia, a highpercentage of newborn cells expressed immature neuronalmarkers Tuj1 and DCX (Fig. 4A and B). At 4 weeks afterhypoglycemia, significantly more new neurons were foundin the GCL of rats subjected to hypoglycemia (40,948BrdU/NeuN double-labeled cells per DG) compared withsham-treated rats (11,860 BrdU/NeuN double-labeled cellsper DG). However, hypoglycemia did not significantly alterthe extent of neuronal differentiation, with 68.2 � 1.51% ofBrdU-labeled cells expressing NeuN at 4 weeks after theinduction of hypoglycemia compared with 66.5 � 1.79% inthe sham group (P � 0.53). In the DG, cells positivelylabeled for both BrdU and GFAP or for both BrdU andCD11b were only detected near the site of injury within theGCL (Fig. 4A); they were otherwise seen in the dentatehilus (data not shown).No neuronal regeneration in the CA1 after hypogly-

cemia. Despite substantial cell loss in the CA1, there was nosignificant increase in the number of CA1 neurons between 2and 4 weeks after the induction of hypoglycemia (Table 1),unlike the increase observed after cerebral global ischemia(17). We again deployed double immunofluorecence stainingwith BrdU and neuronal glial markers to reveal the identity ofproliferating cells in the CA1. In contrast to findings withcerebral global ischemia, no colocalization between BrdUand NeuN or BrdU and Tuj1 was ever detected (Fig. 5A).Most of the cells incorporating BrdU were smaller than theCA1 pyramidal neurons and were colocalized with microglial

marker CD11b (Fig. 5B). Some BrdU cells in the CA1 werealso positive for GFAP marker, suggesting gliogenesis ratherthan neurogenesis in this region.

DISCUSSION

The morphologic and functional sequelae following hypo-glycemic brain injury are well established, but little is

A

FIG. 4. Most of the newborn cells in the GCL assume neuronalphenotypes after hypoglycemia. A: Confocal 3D-reconstruction imagesof newborn cells in the GCL of the DG from rats killed at 2 (DCX, Tuj1,and CD11b) and 4 (NeuN, Calbindin28kd, and GFAP) weeks afterhypoglycemia, showing the neuronal and glial identity of newly dividedcells by BrdU labeling in green, neuronal/glial markers in red, andmerged images. Within the GCL, cells coexpressing BrdU/GFAP andBrdU/CD11b were only seen in areas of injury. Reconstructed orthog-onal images for colocalized cells are presented as viewed in the x–z

(top) and y–z (right) planes. B: Consecutive 0.66–0.7 �m–thick con-focal images from partial z stacks (nos. 1–6) (X630) in green, red, andmerged channels showing colocalization of BrdU and neuronal markers(arrowheads) and cells immunoreactive only for BrdU (arrows). Scalebars, 10 �m.

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FIG. 4—Continued

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FIG. 5. No neuronal regeneration has been detected in the CA1 region of the hippocampus after hypoglycemia. A: Consecutive images fromconfocal partial z stack (nos. 1–5) (X630) in BrdU (green), NeuN and Tuj1 (red), and merged channel showing that the remaining NeuN and Tuj1immunoreactive cells in the CA1 at 4 weeks following hypoglycemia did not colocalized with BrdU (arrows). Despite in close proximity,BrdU-labeled cells (arrows) are on different focal planes from corresponding NeuN- and Tuj1-labeled cells. B: Whereas many of the BrdU-labeledcells were colocalized with either GFAP or CD11b immunoreactive cells (arrowheads) shown in consecutive confocal partial z-stack images (nos.1–6), suggesting that most newborn cells in the CA1 after hypoglycemic brain injury are of glial origin. Cells immunoreactive only for BrdU arelabeled with arrows. Scale bars, 10 �m.

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known about the potential for brain repair and regenera-tion after a hypoglycemic insult. The principal finding ofthis study was that a transient increase in both progenitorcell proliferation and neurogenesis occurred in the SGZand GCL after severe hypoglycemia, followed by a reducedproduction of new neurons below baseline level severalweeks later. In addition to the diminished neurogenesisinduced by hypoglycemia, afterward, the early transientincrease in neurogenesis was insufficient to replace theneuronal loss in the DG, thus resulting in permanentstructural damage. Moreover, unlike findings in rats un-dergoing cerebral global ischemia, no neuronal regenera-tion was detected in the CA1 in the rats subjected tohypoglycemia. Our finding suggests that the pattern ofneuronal regeneration in the hippocampus following hy-poglycemic injury differs from that of cerebral globalischemia, perhaps reflecting the extent and location ofneuronal loss in these two different types of insults.

Neuronal loss was found in the several brain regionsvulnerable to both hypoglycemia and cerebral ischemia(12). However, within each region the damage was notdistributed the same way in the rats subjected to hypogly-cemia as it was in those with cerebral ischemia (12).Infarction or cavitation generally did not occur afterhypoglycemia, whereas they frequently occurred followingischemia. After hypoglycemia, the caudatoputamen wasinvolved more heavily near the white matter, and a super-ficial-to-deep gradient in the density of neuronal loss wascommonly found in the cortex. The hippocampus alsoshowed distinct patterns of neuronal loss at the crest ofthe DG and a gradient of increasing selective neuronal lossmedially in CA1, with marked loss in the subiculum (Fig.2). The unique distribution of neuronal loss followinghypoglycemia is probably related to fluid-born toxin (12).

Adult hippocampal progenitor cells divide at the DGSGZ and produce thousands of new neurons every day inthe GCL (4,5). These new neurons develop the morpho-logical and functional properties of dentate granule cellsand become integrated into existing neuronal circuitries(7,8). Although the function of hippocampal neurogenesisin adult is not yet clear, inhibition of hippocampal neuro-genesis is associated with reduced learning and memorycapacities (9), and impairment of hippocampal neurogen-esis may be linked to cognitive decline in aging, Alzhei-mer’s disease, and major depression (23–25). The factorsinvolved in the regulation of hippocampal neurogenesis inadults are not well defined, but hormones, neurotransmit-ters, environmental stimuli, and growth factors have beenidentified as mediators (26). Pathological conditions in-cluding cerebral ischemia (10,27), seizure (28), Alzhei-mer’s disease (29), and traumatic brain injury (30) areknown to temporarily increase neurogenesis in the DG.Exposure to an enriched environment is linked to im-proved brain function and increased neurogenesis in thehippocampus (31) but not in the olfactory bulb (32), whichfurther supports the unique functional role of hippocampalprogenitor cells. We found that hypoglycemic brain injurystimulated a transitory neurogenesis at the DG (Fig. 3 and4), whereas no significant change in the extent of prolif-eration in the progenitor cells at the SVZ was observed(P � 0.063; mean area of BrdU immunoreactivity:136294.74 �m2 in sham vs. 148397.62 �m2 in hypoglyce-

mia). The difference in the response of progenitor cells tohypoglycemia between the SVZ and SGZ is not wellunderstood. It might be due to the differences in theneurogenic environment between two locations resultingfrom hypoglycemic brain injury.

Recurrent severe hypoglycemia results in permanentcognitive impairment in humans as a consequence ofdamage to the medial temporal lobe (33,34) including thehippocampus (33,35,36). Numerous clinical studies sug-gest that intensive insulin treatment of type 1 diabetes isassociated with an increased frequency of hypoglycemiccoma (37–39) and cognitive impairment (40,41). Youngchildren who have diabetes frequently have memory andcognitive deficits (37), and infants of diabetic mothers alsoshowed significant memory deficits, as evaluated by event-related potential (42,43). These relationships suggest thatthe developing brain is especially vulnerable to hypogly-cemic damage. One possible cause for this phenomenon isthat hypoglycemia damages progenitor cells that are re-quired for brain development. Although there are noavailable data to prove progenitor cell loss in the youngwho have suffered from hypoglycemic brain injury, ourresults in the adult rats with severe hypoglycemia pro-vided convincing evidence to support such a notion (Fig.2A and B). Another mechanism that quite likely contrib-utes to hypoglycemia-associated cognitive dysfunction inboth young and adult human beings is hippocampal neu-ronal loss and insufficient regeneration. Although a recentreport speculated that impaired synaptic plasticity plays arole in cognitive deficits in young rats (16), the basis forthe proposed hypothesis was a model of mild repetitivehypoglycemia that produced no cell death in the hip-pocampus. Nevertheless, the transient increase in neuro-genesis observed in this study was insufficient tocompensate for the neuronal loss that took place inmultiple brain regions, including the DG. In contrast toobservations after cerebral ischemia, no regeneration ofhippocampal CA1 pyramidal neurons was observed in ourmodel of hypoglycemia. Our stereological evaluationshowed that the number of CA1 neurons did not increaseover a period of 4 weeks, as compared with the numberextant after ischemia (17). Confocal Z-stack analysis re-vealed that there were no neuronal markers colocalizedwith BrdU labeling in the CA1 (Fig. 5A), and all prolifer-ating cells in the CA1 proper were either microglia orastroglia (Fig. 5B). The difference in the regenerativepotential of CA1 neurons between our model of hypogly-cemic brain injury and the reported ischemic model mightbe due to the difference in the severity of the CA1 injuries.A �1% survival of neurons in the CA1 after severe isch-emic insult probably triggered the recruitment of progen-itor cells from the posterior periventricular wall toparticipate in CA1 repair (17).

Reduced neurogenesis in the DG seen 4 weeks afterhypoglycemia in this study could be due to loss of progen-itor cells as a result of direct injury to these cells. It couldalso be attributed to a reduced survival of newly formedneuroblasts resulting from an unfavorable microenviron-ment induced by hypoglycemic injury. Hypoglycemiacauses inflammation and microglia activation (44). En-hanced CD11b expression started as early as 2 days,peaked at �1 week, and lasted up to 2 months after the

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induction of hypoglycemia. The spatial distribution ofCD11b expression coincided with areas of neuronal loss,including layer II/III of the cortex, striatum, subiculum,CA1–3, and DG. Activated microglia were also observed incorpus callosum (data not shown). Activated microglia arecytotoxic by releasing reactive oxygen species, proteolyticenzymes, arachidonic acid metabolites, and inflammatorycytokines (45). Neuroinflammation and microglial pathol-ogy are associated with many diseases of cognition, in-cluding Alzheimer’s disease, Lewy body dementia, andAIDS dementia complex (46–48). Lipopolysaccharide-in-duced activated microglia in vitro also produce solubleantineurogenic factors that reduce neuronal differentia-tion into DCX-expressing cells from cocultured neuralprogenitor cells (49). Activated microglia are localized inclose proximity to the newly formed cells, and the impair-ment of neurogenesis depends on the degree of microgliaactivation (50), suggesting that inflammation compromisesthe survival of the new hippocampal neurons. The nonste-roidal anti-inflammatory drug indomethacin restores new-born neurons and reduces activated microglia by 35% inthe DG after cranial irradiation (49), supporting a role forinflammation in inhibiting neurogenesis. Moreover, an-other anti-inflammatory drug, minocycline, also restoredneurogenesis after generalized seizure (50). Based on ourfinding, we speculate that there is a link between chronicinflammation after repeated hypoglycemic injury andfailed neurogenesis. Reduced progenitor cell proliferationand lack of neuronal replacement in many brain regionsmay limit the recovery of cognitive function in patientswith type 1 diabetes. Our results also suggest that it mightbe necessary to augment neurogenesis by prolongingnewborn neuron survival with anti-inflammatory agents inaddition to boosting endogenous repair by growth factorinfusion or behavioral manipulation to combat hypoglyce-mia-induced cognitive impairment.

ACKNOWLEDGMENTS

This work was supported by National Institutes of HealthGrant R01 NS40469 (to J.L.), the Department of VeteransAffairs Merit Program (to J.L.), American Heart Associa-tion Grant SDG003007 (to J.L.), Juvenile Diabetes Re-search Foundation Grant 3-2004-298 (to S.W.S.),Department of Veterans Affairs REAP Grant (to R.A.S.),and Department of Veterans Affairs Merit program (toP.R.W.).

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