Cell Transplantation, Vol. 24, pp. 921–937, 2015 Printed in the USA. All rights reserved. Copyright © 2015 Cognizant Comm. Corp.

0963-6897/15 $90.00 + .00 DOI: http://dx.doi.org/10.3727/096368914X678580 E-ISSN 1555-3892 www.cognizantcommunication.com

Transplantation of Human Dental Pulp-Derived Stem Cells Protects Against Heatstroke in Mice Ling-Shu Tseng,* Sheng-Hsien Chen,†‡§ Mao-Tsun Lin,†§ and Ying-Chu Lin* *School of Dentistry, Kaohsiung Medical University, Kaohsiung City, Taiwan †Department of Medical Research, Chi Mei Medical Center, Tainan City, Taiwan ‡Da-An Women and Children Hospital, Tainan, Taiwan §Department of Biotechnology, Southern Taiwan University of Science and Technology, Tainan, Taiwan

Stem cells from human exfoliated deciduous tooth pulp (SHED) is a promising approach for the treatment of stroke and spinal cord injury. In this study, we investigated the therapeutic effects of SHED for the treatment of multiple organ (including brain, particularly hypothalamus) injury in heatstroke mice. ICR male mice were exposed to whole body heating (WBH; 41.2°C, relative humidity 50–55%, for 1 h) and then returned to normal room temperature (26°C). We observed that intravenous administration of SHED immediately post-WBH exhibited the following therapeutic benefits for recovery after heatstroke: (a) inhibition of WBH-induced neurologic and thermoregulatory deficits; (b) reduction of WBH-induced ischemia, hypoxia, and oxidative damage to the brain (particularly the hypothalamus); (c) attenuation of WBH-induced increased plasma levels of systemic inflammatory response molecules, such as tumor necrosis factor-a and intercellular adhesion molecule-1; (d) improvement of WBH-induced hypothalamo–pituitary–adrenocortical (HPA) axis activity (as reflected by enhanced plasma levels of both adrenocorticotrophic hormone and corticosterone); and (e) attenuation of WBHinduced multiple organ apoptosis as well as lethality. In conclusion, post-WBH treatment with SHED reduced induction of proinflammatory cytokines and oxidative radicals, enhanced plasma induction of both adrenocorticotrophic hormone and corticosterone, and improved lethality in mouse heatstroke. The protective effect of SHED may be related to a decreased inflammatory response, decreased oxidative stress, and an increased HPA axis activity following the WBH injury. Key words: Heatstroke; Human exfoliated deciduous teeth pulp stem cells; Brain; Thermoregulation; Neurologic severity scores; Multiple organs

INTRODUCTION Heatstroke-induced deaths are increasing with global warming and with a worldwide increase in the frequency and intensity of heat waves (28). In terms of the clinical burden on society, heatstroke-induced brain injury is the third largest killer in the world after cardiovascular disease and traumatic brain injury (28,29). Heatstroke is characterized by hyperthermia, systemic inflammatory response syndromes, multiple organ failure, and brain dysfunction (3,7). Environmental heat stress increases cutaneous blood flow and metabolism and progressively decreases splanchnic blood flow. Severe heat stress also decreases mean arterial pressure, increases intracranial pressure, and results in decreased cerebral perfusion pressure, which leads to cerebral ischemia and hypoxia. Compared with normothermic controls, rodents with heatstroke have more cellular levels indicative of ischemia and damage and prooxidant enzymes in the brain, as well

as more neuronal damage. In contrast, people suffering from heatstroke have fewer antioxidants in the brain. Stem cells from human exfoliated deciduous teeth (SHED) are self-renewing stem cells residing within the perivascular niche of the dental pulp (12). SHED can be obtained without adverse health effects because naturally exfoliated deciduous teeth are not needed. Like bone marrow mesenchymal stem cells, these cells are able to differentiate into functionally active neurons in vitro under certain circumstances (1,35). Trophic factors expressed by them promote neuronal survival (2,15). SHED are thought to originate from the cranial neural crest and express early markers for both mesenchymal and neuroectodermal stem cells (1,21). It has been shown that human dental pulp-derived stem cells protect against stroke (16,36) and spinal cord injury (26) in rodents. However, whether engrafted SHED offer therapeutic benefits in heatstroke is still unknown.

Received July 16, 2013; final acceptance January 29, 2014. Online prepub date: March 7, 2014. Address correspondence to Dr. Ying-Chu Lin, Ph.D., School of Dentistry, Kaohsiung Medical University, Kaohsiung City 807, Taiwan. Tel: 866 7 3121101, ext. 2753; Fax: 866 7 3157024; E-mail: [email protected]

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Here we examined the protective activities of SHED by transplanting them into mice subjected to heatstroke, in which brain (hypothalamic) ischemia and oxidative damage, multiple organ dysfunction, and lethality can be evaluated accurately. Our data show that SHED displayed beneficial effects in treating experimental heatstroke. MATERIALS AND METHODS SHED Harvest From Deciduous Teeth Human SHED were isolated as described previously (21). Exfoliated deciduous teeth (from males and females 6–12 years old) extracted for clinical purposes were collected under approved guidelines from the Institutional Review Board of Chi Mei Medical Center (Tainan, Taiwan), and informed consent (parent/guardian) was obtained. After separation of the crown and root, the dental pulp was isolated and then digested in a solution of 3 mg/ml collagenase (Sigma, St. Louis, MO, USA) type 1 and 4 mg/ml dispase (Sigma) for 1 h at 37°C. After infiltration through 70-µm cell strainers (Talcon; BD Labware, BD Biosciences, Bedford, MA, USA), the cells were cultured at 37°C under 5% CO2 in Dulbecco’s modified Eagle’s medium (Gibco, Carlsbad, CA, USA) containing 20% mesenchymal cell growth supplement (Lonza, Inc., Walkersville, MD, USA) and antibiotics (100 U/ml penicillin, 100 mg/ml streptomycin, and 0.25 mg/ml amphotericin B; Gibco). After primary culture, the cells were subcultured at ~1 × 104 cells/cm2 and used in experiments after three to five passages. All protocols were approved by the Animal Ethics Committee of the Chi Mei Medical Center (Tainan, Taiwan) in accordance with the Guide for the Care and Use of Laboratory Animals and the guidelines of the Animal Welfare Act of the National Science Council of the Republic of China (Taipei, Taiwan). Single-cell suspensions of 1 × 105/0.3 ml of SHED were administered via the tail vein immediately after the termination of whole body heating (WBH). Murine Model of Heatstroke Institute of Cancer Research (ICR) of the National Institutes of Health (Bethesda, MD, USA) mice were purchased from the National Animal Center (Taipei, Taiwan) and kept under a 12-h light–dark cycle at controlled temperature (22 ± 2°C) with free access to food and tap water. ICR male mice 8 to 10 weeks old were exposed to WBH (41.2°C, relative humidity 50–55%, for 1 h) in an environmental temperature-controlled chamber (5,6,9). The heated mice were returned to the normal room temperature (26°C) after the end of WBH. Mice that survived to day 4 of WBH were considered survivals, and the data were used for analysis of the results. In separate experiments, 4 h post-WBH, all of the animals were sacrificed, and their organs were removed for histological and biochemical evaluation. The contents of nitric oxide metabolites (NOx), 2,3-dihydroxybenzoic acid (2,3-DHBA), glutamate, lactate-to-pyruvate ratio, glycerol,

malondialdehyde, oxidative-form glutathione (GSSG), reduced-form glutathione (GSH), glutathione peroxidase (GP), and glutathione reductase (GR) were determined. In addition, numbers of apoptotic cells in the brain (hypothalamus), kidney, spleen, lung, and liver were assessed. For rectal temperature measurements, unrestrained, unanesthetized mice were used, and measurements were collapsed into 10-min averages, taking one mouse from each group and changing the sequence thereafter. Rectal temperatures were measured by a thermocouple probe (Bailey Instruments, Saddlebrook, NJ, USA). Assessment of Thermoregulatory Function Immediately after the termination of WBH, the animals were returned to a room temperature of 26°C for recovery. According to the findings of Chatterjee et al. (5,6), WBH-treated mice were unable to maintain their normal thermoregulation. When they were exposed to room temperature (26°C), they displayed thermoregulatory deficits (e.g., hypothermia). Modified Neurologic Severity Scores (mNSS) Neurologic function was graded on a scale of 0 to 14 (normal score 0; maximal deficit score 14) (18). mNSS is a composite of motor, reflex, and balance tests. In the severity scores of injury, one score is awarded for the inability to perform the test or for the lack of a tested reflex; thus, the higher the score, the more severe is the injury. Neuronal Damage Score At the end of the experiments, animals were sacrificed by an overdose of sodium pentobarbital, and the brains were fixed in situ and left in the skull in 100% neutral-buffered formalin (Sigma) for at least 24 h before removal. The brain was removed and embedded in paraffin blocks. Serial sections (100 mm thick) through the hypothalamus were stained with hematoxylin and eosin (H&E; Sigma) for microscopic examination. The extent of neuronal damage was scored on a scale of 1 to 3, modified from the grading system of Pulsinelli et al. (25), in which 0 is normal, 1 indicates approximately 30% of the neurons are damaged, 2 indicates that approximately 60% of the neurons are damaged, and 3 indicates that 100% of the neurons are damaged. Each hemisphere was evaluated independently by an examiner blinded to the experimental conditions. Assessment of Cerebral Blood Flow and PO2 A 100-µm diameter thermocouple and two 230-µm fibers were attached to the oxygen probe. This combined probe measured oxygen, temperature, and microvascular blood flow. This measurement required OxyLite™ and OxyFlo™ instruments. OxyLite 2000 (Oxford Optronix Ltd., Oxford,

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UK) is a two-channel device (measuring PO2 and temperature at two sites simultaneously), whereas OxyFlo 2000 is a two-channel laser Doppler perfusion-monitoring instrument. Under anesthesia, the mouse was placed in a stereotaxic apparatus, and the combined probe was implanted into the brain (or the hypothalamus) using the atlas and coordinates of Paxinos and Watson (24). Extracellular Levels of Glutamate, Lactate-to-Pyruvate Ratio, Glycerol, Nitric Oxide, and Hydroxyl Radicals in the Brain (or Hypothalamus) Brain (hypothalamus) samples were homogenized in 0.05 M phosphate buffer (Sigma), pH 7.0, and then centrifuged at 4,000 × g for 20 min at 4°C. The supernatants were used for determination of cellular levels of glutamate, lactate-to-pyruvate ratio, glycerol, nitric oxide, and hydroxyl radicals. The dialysis probe (4 mm in length CMA/12; Carnegie Medicine, Stockholm, Sweden) was put into the supernatants to obtain the dialysates. The nitric oxide (NOx−) concentration in the dialysates of hypothalamus was measured with the Eicom ENO-20 NOx− analysis system (Eicom, Kyoto, Japan) (33). In the Eicom ENO-20 NOx− analysis system, after the NO2− and NO3− in the sample have been separated by the column, the NO2− reacts in the acidic solution with the primary aromatic amine to produce an azo compound. Following this, the addition of aromatic amines to the azo compound results in a coupling that produces a diazo compound, and the absorbance rate of the red color in this compound is then measured. For measurement of glutamate, lactate-to-pyruvate ratio, and glycerol, the dialysates were injected onto a CMA600 microdialysis analyzer (Carnegie Medicine). The concentrations of hydroxyl radicals were measured by a modified procedure based on the hydroxylation of sodium salicylates by hydroxyl radicals, leading to the production of 2,3-DHBA and 2,5-DHBA (13). Determination of Lipid Peroxidation Lipid peroxidation was assessed by measuring the levels of brain (or hypothalamus) malondialdehyde (MDA) with 2-thiobarbituric acid (TBA) (Sigma) to form a chromophore absorbing at 532 nm (34). About 0.1 g of tissue was homogenized with 1.5 ml of 0.1 M phosphate buffer at pH 3.5. The reaction mixture (Sigma) (0.2 ml of sample, 1.5 ml of 20% acetic acid, 0.2 ml of 8.1% sodium dodecyl sulfate, and 1.5 ml of aqueous solution of 0.8% TBA, up to 4 ml with distilled water) was heated to 95°C for 1 h, and then 5 ml of N-butanol (Sigma) and pyridine (15:1 v/v) (Sigma) was added. The mixture was vortexed vigorously, centrifuged at 1,500 × g for 10 min, and the absorbance of the organic phase was measured at 532 nm on a microplate spectrophotometer (Thermo Scientific, Waltham, MA, USA). The values were expressed as nanomoles of TBA-reactive substances (MDA equivalent) per milligram of protein.

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Quantification of Total and Oxidized Glutathione Tissues were homogenized in 5% 5-eslfosalicylic acid (1:10 w/v) (Sigma) at 0°C, and the supernatants were used for analysis of total and oxidized glutathione. Total glutathione [reduced-form glutathione (GSH) + oxidized-form glutathione (GSSG)] was analyzed according to the Tieze method (32), and GSSG was determined as described by Griffith (11). The recycling assay for total glutathione is oxidized by 5,5-Dithiosis [2 acid] (DTNB) (Sigma) to give GSSG with stoichiometric formation of 5-thio-2nitro-benzoic acid. GSSG is reduced to GSH by the action of the highly specific GR and nicotinamide adenine dinucleotide phosphate (reduced form; NADPH). The rate of 5-thio-2-nitro-benzoic acid formation is followed at 412 nm and is proportional to the sum of GSH and GSSG present. Determination of Glutathione Peroxidase (GP) and Glutathione Reductase (GR) Activity Tissues were homogenized in 0.05 M phosphate buffer (Sigma), pH 7.0, and then centrifuged at 4,000 × g for 20 min at 4°C. The supernatants were used for GP and GR activity assay. The GP and GR activities were performed with a commercial GP assay kit (Sigma) and a GR assay kit (Sigma), respectively. One unit of GP and GR activity was defined as the amount of sample required to oxidize 1 mmol of NADPH per minute based on the molecular absorbance of 6.22 × 106 for NADPH. Plasma Concentrations of Inflammatory and Intercellular Adhesion Molecules and Cytokines Blood samples were taken at 4 h after the start of heat exposure for determination of tumor necrosis factor-a (TNF-a), interleukin-10 (IL-10), and intercellular adhesion molecule-1 (ICAM-1) levels. The amounts of the cytokines including TNF-a, IL-10, and ICAM-1 in serum were determined by double antibody sandwich enzyme-linked immunoabsorbent assay (R&D Systems, Minneapolis, MN, USA) according to the manufacturer’s instructions. Optical densities were read on a phate reader (Thermo Scientific, Rockford, IL, USA) set at 450 nm for TNF-a, IL-10, and ICAM-1. The concentrations of TNF-a and ICAM-1 in the serum samples were calculated from the standard curve multiplied by the dilution factor and were expressed as picograms per milliliter (19). Plasma Assessment of Adrenocorticotrophic Hormone (ACTH) and Corticosterone Plasma ACTH and corticosterone were assayed using ACTH (Rat, Mouse)-RIA kit (Phoenix Pharmaceuticals, Burlingame, CA, USA) and Corticosterone Double Antibody RIA kit (MP Biomedicals, Solon, OH, USA), respectively. All analyses were performed according to the manufacturer’s instructions.

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TUNEL Assay for Apoptotic Cells An in situ apoptosis detection kit (Clontech, Mountain View, CA, USA) was employed to assess apoptosis by using the terminal deoxynucleotidyl transferase (TdT)mediated dUTP nick-end labeling (TUNEL) method. Four hours after the end of WBH, mice were given an overdose of urethane (1.4 g/kg body weight, IP; Sigma) and then perfused and prefixed with phosphate-buffered saline (PBS) (Sigma) and 10% formaldehyde (Sigma). The brain, lungs, spleen, and kidneys were excised and postfixed in a solution containing 30% sucrose and 10% formaldehyde for at least 24 h. After they had been fixed, the organs were separately embedded in medium (Tissue Tek OCT; Miles Laboratories, Elkart, IN, USA). Snap-frozen samples were cryostat sectioned (8 µm thick) and placed on slides (Sigma) coated with poly-L-lysine for TUNEL assays. In brief, tissue slides were pretreated with 20 µg/ml of proteinase K (Sigma) solution for 5 min and then incubated with the reaction mixture (Sigma) containing TdT and fluorescein-conjugated deoxyuridine triphosphate (dUTP) (Sigma) for 1 h at 37°C. After they had been incubated, the sections were washed with PBS, their nuclei were costained with 4,6-diamidino-2-phenylindole (DAPI) using DAPI-containing mounting medium (Vectashield R; Vector Laboratories, Burlingame, CA, USA) and subsequently analyzed using a fluorescent microscope (E800; Olympus, Tokyo, Japan) equipped with a digital camera (Coolpix995, Olympus). Apoptosis induction efficacy was calculated as a percentage of fluorescein-positive to DAPI-stained nuclei. Cell count was conducted by an investigator who was blinded to the experimental groups. Unbiased stereology was used in the cell counting. Estimated total numbers of neurons within the brain region of interest were obtained by calculating the product of the mean neuron density within the unbiased virtual counting spaces and the global volume of the brain region of interest. Statistical Analysis All values in the figures and text are expressed as mean ± SEM of n observations, where n represents the number of animals studied. Statistical evaluation was performed by using a one-way ANOVA followed by a

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multiple-comparison test (Scheffe’s test). The Kaplan– Meier analysis was used for determining the significant differences in the survival rate between control and treatment groups. The Wilcoxon tests were used for evaluation of neuronal damage scores. The Wilcoxon test converts the scores or values of a variable to ranks, requires calculation of a sum of the ranks, and provides critical values for the sum necessary to test the null hypothesis at a given significance level. These data were presented as “median,” followed by first (Q1) and third (Q3) quartile. A value of p < 0.05 was considered to be statistically significant. Statistica 12.0 (Stat Soft Holdings, Inc., Taiwan Branch, New Taipei City, Taiwan) was used to analyze all data. RESULTS Thermoregulatory and Neurologic Outcome and Lethality by WBH Functional tests showed balanced thermoregulatory and neurological deficits between heated control and heated SHED-treated groups. WBH mice treated with SHED showed significant (p < 0.05, F < 3.49, n = 12/group) improvement of functional recovery on thermoregulatory test at 4–16 h and mNSS at 12–48 h compared with vehicle-treated mice, respectively (Fig. 1). The mortality rate of SHED-treated heated mice was 10 of 12 mice, and 1 of 12 heated control mice (p < 0.05, F < 5.95) (Fig. 1). Cerebral Cell Damage and Apoptosis by WBH Histopathological verification showed that hypothalamus values of cell damage scores (Table 1, Fig. 2), TUNEL-positive cells (Fig. 3), and caspase 3-positive cells (Fig. 4) for vehicle-treated heated mice were all significantly (p < 0.05, F < 3.49) higher 4 h post-WBH than they were for the nonheated control mice. Vehicle-treated WBH mice displayed cell body shrinkage, pyknosis of the nucleus, loss of Nissl substance, disappearance of the nucleolus (Fig. 2), and apoptotic changes (Figs. 3 and 4). Compared to nonheated control mice, vehicle-treated WBH mice also had significantly (p < 0.05, F < 3.49) higher levels of cellular damage markers (e.g., glycerol)

FACING PAGE Figure 1. Thermoregulatory dysfunction, neurologic deficit, and lethality by WBH. (A) Thermoregulatory dysfunction [or animals become hypothermic when exposed to room temperature (26°C) caused by WBH (41.2°C for 1 h)]. Data are expressed as means ± SEM of 12 mice per group. *At time 0 h post-WBH (or immediately after the termination of WBH), WBH mice treated with vehicle (WBH + vehicle; ○) or WBH mice treated with SHED (1 × 105/0.3 ml; ▿) had significantly higher rectal temperature (~42.5°C) than those of non-WBH mice treated with vehicle (non-WBH + vehicle; ●). In contrast, at time 4, 8, 12, or 16 h post-WBH, WBH + vehicle mice had significantly lower rectal temperatures (~36–33°C) than those of non-WBH + vehicle mice or WBH + SHED mice (p < 0.05; F < 3.49). (B) Neurologic deficit (or decreased mNSS scores) caused by WBH. Data are expressed as means ± SEM of 12 mice per group. *p < 0.05, F < 3.49, WBH + vehicle (▨) versus non-WBH + vehicle (□); +p < 0.05, F < 3.49, WBH + SHED (▩) versus WBH + vehicle (▨). (C) Lethality (or decreased percentage survival) caused by WBH. *p < 0.01, F < 5.95, non-WBH + vehicle mice versus WBH + vehicle mice or WBH + SHED mice versus WBH + vehicle.

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Table 1. Effects of Heat Exposure on Neuronal Damage Score of the Hypothalamus in Different Groups of Mice Treatment Groups

Neuronal Damage Score (0–3)

1. Non-WBH mice 2. WBH mice treated with vehicle saline (0.3 ml, IV) 3. WBH mice treated with SHED (1 × 105 cells/0.3 ml, IV)

0 (0, 0) 2 (2, 2)* 0.75 (0, 0.75)†

Samples were measured 4 h post-WBH (41.2°C for 1 h) or the equivalent time period for the nonheated group. *Compared with non-WBH group (p < 0.01; F < 5.95); †compared with group 2 (p < 0.05; F < 3.49). Data are means ± SEM of 12 mice per group.

(4,14) (Table 2). SHED-treated heated mice show significant (p < 0.01, F < 5.95) improvement of cerebral apoptosis and damage (Tables 1 and 2). Cerebral Ischemia and Hypoxia by WBH Intracerebral assessment revealed that cerebral levels of both cerebral blood flow and PO2 for vehicle-treated heated mice were significantly (p < 0.01, F < 5.95) higher at 4 h post-WBH than they were for the nonheated control mice (Table 1). Again, the cerebral levels of cellular ischemia markers (e.g., glutamate and lactate/pyruvate ratio) (4,14) for vehicle-treated heated mice were significantly (p < 0.05, F < 3.49) higher at 4 h post-WBH than they were for the nonheated control mice (Table 2). Heated mice treated with SHED showed significant (p < 0.01, F < 5.95) improvement of cerebral ischemia and hypoxia (Table 2). Oxidative Stress by WBH Biochemical determination showed that hypothalamic levels of MDA, GSSG/GSH, NOx−, and 2,3-DHBA for vehicle-treated heated mice were all significantly (p < 0.01, F < 5.95) higher 4 h post-WBH than they were for the nonheated control mice (Table 3). In contrast, hypothalamic levels of GP and GR for vehicle-treated heated mice were significantly (p < 0.01, F < 5.95) lower than they were for the nonheated control mice (Table 3). Heated mice treated with SHED showed significant (p < 0.01, F < 5.95) improvement of oxidative stress caused by WBH (Table 3). Increased Plasma Levels of both ACTH and Corticosterone by WBH Biochemical verification showed that plasma levels of both ACTH and corticosterone for vehicle-treated heated mice were significantly (p < 0.05, F < 3.49) higher 4 h post-WBH than they were for the nonheated control mice (Table 4). Heated mice treated with SHED showed significant (p < 0.05, F < 3.49) enhancement of both ACTH and corticosterone in plasma by WBH (Table 4).

Figure 2. Brain H&E staining 4 h post-WBH. Photographs showing hypothalamic H&E staining for a non-WBH + vehicle mouse, a WBH + vehicle mouse, and a WBH + SHED mouse. Samples were obtained 4 h post-WBH for the WBH groups or the equivalent time period for the non-WBH group. WBH + vehicle mice showed cell body shrinkage, pyknosis of the nucleus, loss of Nissl substance, and disappearance of the nucleolus, which could be attenuated by SHED treatment (please see arrows).

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Figure 3. Hypothalamus TUNEL staining 4 h post-WBH. Values of TUNEL-positive cells in the brain section for the non-WBH + vehicle mice (□), WBH + vehicle mice (▨), and WBH + SHED mice (▩). Samples were obtained 4 h post-WBH for the WBH groups or the equivalent time period for the non-WBH group. Data were means ± SEM of six animals per group. *p < 0.05, F < 3.49 compared to □ group; +p < 0.05, F < 3.49 compared to ▨ group. Photographs showing brain TUNEL staining for a nonWBH mouse, a WBH + vehicle mouse, and a WBH + SHED mouse. Please see the legend of Figure 1 for the abbreviations.

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Figure 4. Hypothalamus caspase 3 staining 4 h post-WBH. Values of caspase 3-positive cells in the brain section for the nonWBH + vehicle mice (□), WBH + vehicle mice (▨), and WBH + SHED mice (▩). Samples were obtained 4 h after the start of WBH (41.2°C for 1 h) for the WBH groups or the equivalent time period for the non-WBH group. *p < 0.05, F < 3.49 compared to (□) group; +p < 0.05, F < 3.49 compared to (▨) group. Photographs showing hypothalamic caspase 3 staining for a NC mouse, a HS + saline mouse, a HS + HUCBC mouse, and a HS + SHED mouse. Please see the legend of Figure 1 for the abbreviations.

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Table 2. Effects of Heat Exposure on Hypothalamic Levels of Glutamate, Lactate/Pyruvate, Glycerol, Cerebral Blood Flow (CBF), and PO2 in Different Groups of Mice Treatment Groups

Glutamate (% of Baseline)

Lactate/Pyruvate Ratio

Glycerol (% of Baseline)

CBF (BPU)

PO2 (mmHg)

100 ± 5 213 ± 12*

11 ± 2 224 ± 16*

100 ± 6 189 ± 9*

348 ± 22 162 ± 17*

20 ± 3 6 ± 2*

114 ± 3†

13 ± 3†

101 ± 5†

285 ± 19†

14 ± 2†

1. Non-WBH mice 2. WBH mice treated with vehicle saline (0.3 ml, IV) 3. WBH mice treated with SHED (1 × 105 cells/0.3 ml, IV)

Samples were measured 4 h post-WBH or the equivalent time period for nonheated group. *Compared with non-WBH group (p < 0.01; F < 5.95); †compared with group 2 (p < 0.05; F < 3.49). Data are means ± SEM of 12 mice per group.

Multiple Organ Apoptosis by WBH Immunohistochemical verification showed that numbers of the TUNEL-positive cells in the kidney (Fig. 5), the spleen (Fig. 6), the lung (Fig. 7), and the liver (Fig. 8) for the vehicle-treated heated mice were significantly (p < 0.05, F < 3.49) higher 4 h post-WBH than they were for the nonheated control mice. Heated mice treated with SHED showed significant (p < 0.05, F < 3.49) improvement of apoptosis in multiple organs (Figs. 5–8). Increased Serum Levels of Systemic Inflammatory Response Indicators by WBH Biochemical determination showed that serum levels of several systemic inflammatory response indicators, such as TNF-a and ICAM-1, for vehicle-treated heated mice were significantly (p < 0.05, F < 3.49) higher 4 h post-WBH than they were for the nonheated mice (Fig. 9). Heated mice treated with SHED showed significant (p < 0.05, F < 3.49) reduction of the increased serum levels of these two systemic inflammatory response indicators by WBH (Fig. 9). In addition, the serum levels of IL-10, an antiinflammatory cytokine, were further significantly (p < 0.05, F < 3.49) increased following SHED therapy (Fig. 9). DISCUSSION We report here that intravenous administration of SHED exhibits the following therapeutic benefits for recovery of heat stroke: (a) inhibition of WBH-induced neurologic and

thermoregulatory deficits; (b) reduction of WBH-induced ischemic, hypoxic, and oxidative damage to the hypothalamus; (c) attenuation of WBH-induced plasma overproduction of systemic inflammatory response molecules; (d) reduction of WBH-induced hypothalamo–pituitary– adrenocortical (HPA) axis impairment as reflected by increasing plasma levels of both adrenocorticotrophic hormone and corticosterone; and (e) attenuation of WBH-induced lethality. Post-WBH treatment with SHED improved functional and histological outcomes of multiple organs (including brain or hypothalamus) and reduced induction of proinflammatory cytokines and oxidative radicals in heatstroke mice. The protective effect of SHED may be related to a decreased inflammatory response as well as an increase in the HPA axis activity following the injury. Adult mesenchymal stem cells have been isolated from various tissues, including bone marrow, adipose tissue, skin, umbilical cord, and placenta (17,26,37,38). Previous studies demonstrated that intravenous delivery of human umbilical cord blood-derived stem cells resuscitated the anesthetized rats with heatstroke by reducing circulatory shock and cerebral ischemia (7). The present results further showed that SHED protected against heatstroke in unanesthetized mice. In conclusion, SHED attenuated systemic inflammation, ameliorated apoptosis of multiple organs (including the brain), and promoted thermoregulatory and neurological recovery and survival in heat stroke mice. SHED have significantly higher proliferation rates

Table 3. Effect of Heat Exposure on Hypothalamic Levels of MDA GSSG/GSH, GP, GR, NO x−, and 2,3-DHBA in Different Groups of Mice Treatment Groups 1. Non-WBH mice 2. WBH mice treated with vehicle saline 3. WBH mice treated with SHED (1 × 105/0.3 ml, IV)

MDA GP GR (nmol/mg Protein) GSSG/GSH (mU/mg Protein) (mU/mg Protein)

NOx−

2,3-DHBA (% of Baseline)

4±1 11 ± 3*

0.49 ± 0.15 2.36 ± 0.37*

303 ± 35 80 ± 16*

179 ± 17 77 ± 12*

18 ± 2 102 ± 11*

100 ± 7 171 ± 9*

4 ± 1†

0.44 ± 0.14†

244 ± 29†

151 ± 17†

20 ± 3†

102 ± 6†

Samples were measured 4 h post-WBH or the equivalent time period for non-WBH group. *Compared with non-WBH group (p < 0.01; F < 5.95); †compared with group 2 (p < 0.01; F < 5.95). Data are means ± SEM of 12 mice per group.

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Table 4. Effect of Heat Exposure on Plasma Levels of Both ACTH and Corticosterone for Various Groups of Mice Treatment Groups 1. Non-WBH mice 2. WBH mice treated with vehicle saline 3. WBH mice treated with SHED (1 × 105/0.3 ml, IV)

ACTH (pg•ml−1)

Corticosterone (ng•ml−1)

395 ± 83 624 ± 123* 1,872 ± 156†

136 ± 22 247 ± 24* 668 ± 31†

Samples were measured 4 h post-WBH or the equivalent time period for non-WBH group. *Compared with non-WBH group (p < 0.05; F < 3.49); †compared with group 2 (p < 0.05; F < 3.49). Data are means ± SEM of 12 mice per group.

than that of bone marrow mesenchymal stem cells and have the added advantages of being simple to harvest and express several growth factors (22). Dental pulp stem cells ameliorated ischemic tissue injury in the rat brain and accelerated functional recovery after middle cerebral artery occlusion (MCAO) in one study (31). These cells released vascular endothelial growth factor and promoted the migration and differentiation of endogenous neuronal progenitor cells (NPCs) in the subventricular zone (31). However, SHED cell therapy is associated with several problems, such as limited amount of SHED, tumorigenesis, immune rejection, and ethical issues (16,36). Studies in recent years have suggested that stem cell transplantation may be regarded as cell-based cytokine therapy (23,31). Indeed, several studies have reported that growth factors derived from transplanted stem cells accelerate the recovery of several diseases, including focal cerebral ischemia (31), amyotrophic lateral sclerosis (23), and traumatic brain injury (10) in rats. SHED-derived conditioned medium promoted the migration and differentiation of endogenous NPCs, induced vasculogenesis, and ameliorated ischemic brain injury after MCAO (16). Additionally, SHED graft and SHED-conditioned medium improved neurological outcome, inhibited apoptosis, and reduced brain tissue loss in neonatal mice with brain ischemic–hypoxic injury (35). Furthermore, Sakai and colleagues (27) demonstrated that tooth-derived stem cells protected against spinal cord injury through both cell-autonomous and paracrine neuroregenerative activities. Although it was difficult to compare the level of therapeutic benefits between engrafted SHED and SHED-conditioned medium, cell transplantation may have an advantage in providing a prolonged delivery of paracrine factors, compared with the transient delivery by the conditioned medium treatment (36). In the present study, we only showed the phenotype of different treatment groups. Different animal models and different niches may influence the production and dynamics of growth factors from SHED. In this way, SHED may not necessarily exhibit the same features of other sources of stem cells. Further studies are warranted to prove the precise mechanism or production of SHED-mediated growth factors in our heatstroke model. According to a recent review (8), ischemic and oxidative damage to the hypothalamus may be responsible for

heatstroke. Indeed, as shown in the present results, mice displayed profound hypothermia at room temperature exposure 4 h post-WBH, indicating thermoregulatory dysfunction. Thermoregulatory deficits after heatstroke in mice can be explained by the inflammatory, ischemic, and oxidative damage to the hypothalamus. The contention is in part supported by previous findings showing that decreased heat tolerance is associated with HPA axis impairment in rats (20). Severe heat stress increases cutaneous blood flow and metabolism and decreases splanchnic blood flow. In addition, severe heat stress decreases mean arterial pressure, increases intracranial pressure, and decreases cerebral perfusion pressure, all of which lead to cerebral ischemia and hypoxia. Compared with normothermic controls, rodents with heatstroke have higher values of cerebral ischemia markers (e.g., increased glutamate and lactate-to-pyruvate ratio) and damage (e.g., increased glycerol), prooxidant enzyme markers (e.g., increased lipid peroxidation and glutathione oxidase), proinflammatory cytokines (e.g., increased IL-1b, TNF-a, and ICAM-1), nitric oxide, an indicator for the accumulation of polymorphonuclear leukocytes (e.g., myeloperoxidase activity), and neuronal degeneration (e.g., apoptosis) in the hypothalamus after heatstroke. Brain or hypothalamic values of antioxidant defenses (e.g., increased GP and GR), however, are lower. The ischemic, inflammatory, and oxidative damage to the hypothalamus during heatstroke may cause multiple organ dysfunction or failure through HPA axis mechanisms. Indeed, as shown in the present results, WBH-treated mice with vehicle treatment presented the profound hypothermia and remarkable cerebral ischemia, hypoxia, oxidative stress, and neuronal damage, but the lowest plasma ACTH and corticosterone levels. Conversely, WBH-treated mice with SHED treatment showed a lesser mNSS score, a slight hypothermia, and a lesser ischemia, hypoxia, oxidative stress, and brain cell damage, but higher plasma levels of both ACTH and corticosterone. Systemic inflammatory response syndrome molecules, including TNF-a and ICAM-1, are increased in heatstroke patients (3) and rats (19). Our present results further showed that increased serum levels of these systemic inflammatory response syndrome molecules in heatstroke mice can be significantly attenuated following SHED therapy.

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Figure 5. Renal TUNEL staining 4 h post-WBH. Values of TUNEL-positive cells in the kidney section for the non-WBH mice (□), WBH + vehicle mice (▨), and WBH + SHED mice (▩). Samples were obtained 4 h after the start of WBH (41.2°C for 1 h) for the WBH groups or the equivalent time period for the non-WBH group. *p < 0.05, F < 3.49 compared to □ group; +p < 0.05, F < 3.49 compared to ▨ group. Photographs showing kidney TUNEL staining for a non-WBH mouse, a WBH + vehicle mouse, and a WBH + SHED mouse. Please see the legend of Figure 1 for the abbreviations.

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Figure 6. Splenic TUNEL staining 4 h post-WBH. Values of TUNEL-positive cells in the spleen section for the non-WBH + vehicle mice (□), WBH + vehicle mice (▨), and WBH + SHED mice (▩). Samples were obtained 4 h after the start of WBH (41.2°C for 1 h) for the WBH groups or the equivalent time period for the non-WBH group. *p < 0.05, F < 3.49 compared to □ group; +p < 0.05, F < 3.49 compared to ▨ group. Photographs showing spleen TUNEL staining for a non-WBH mouse, a WBH + vehicle mouse, and a WBH + SHED mouse. Please see the legend of Figure 1 for the abbreviations.

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Figure 7. Pulmonary TUNEL staining 4 h post-WBH. Values of TUNEL-positive cells in the lung section for the non-WBH mice (□), WBH + vehicle mice (▨), and WBH + SHED mice (▩). Samples were obtained 4 h post-WBH for the WBH groups or the equivalent time period for the non-WBH group. Data were means ± SEM of six animals per group. *p < 0.05, F < 3.49 compared to □ group; +p < 0.05, F < 3.49 compared to ▨ group. Photographs showing pulmonary TUNEL staining for a non-WBH mouse, a WBH + vehicle mouse, and a WBH + SHED mouse. Please see the legend of Figure 1 for the abbreviation.

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Figure 8. Hepatic TUNEL staining 4 h post-WBH. Values of TUNEL-positive cells in the liver section for the non-WBH mice (□), WBH + vehicle mice (▨), and WBH + SHED mice (▩). Samples were obtained 4 h post-WBH for the WBH groups or the equivalent time period for the non-WBH group. Data were means ± SEM of six animals per group. *p < 0.05, F < 3.49 compared to □ group; +p < 0.05, F < 3.49 compared to ▨ group. Photographs showing hepatic TUNEL staining for a non-WBH mouse, a WBH + vehicle mouse, and a WBH + SHED mouse. Please see the legend of Figure 1 for the abbreviations.

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Figure 9. Serum levels of cytokines 4 h post-WBH. Values of serum levels of TNF-a, IL-10, and ICAM-1 for the non-WBH mice (□), WBH + vehicle mice (▨), and WBH + SHED mice (▩). Samples were obtained 4 h after the start of WBH (41.2°C for 1 h) for the WBH groups or the equivalent time period for the non-WBH group. *p < 0.05; F < 3.49 , compared to □ group; +p < 0.05, F < 3.49 compared to ▨ group.

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The serum levels of an anti-inflammatory cytokine, IL-10 (30) was increased following stem cell therapy after heatstroke. These observations indicate that heat-induced activated inflammation in mice can be reduced by SHED treatment. Finally, it should be mentioned that the antiapoptotic effect provided by not only SHED but also by other sources of stem cells may also provide the suppression of inflammatory cytokines and reduction of oxidative damage. This study suggests that intravenous administration of SHED may help in the recovery of acute heatstroke in patients in the future. In conclusion, protective therapy using SHED is a potential candidate for innovative therapy for heatstroke since it is very safe with no associated problems. ACKNOWLEDGMENTS: This study was funded in part by the National Science Council of the Republic of China (Grant Nos. NSC100-2314-B-218-001 and NSC102-2628-B-384-001MY3) and the Department of Health of the Republic of China (DOH99-TD-B-111-003, Center of Excellence for Clinical Trial and Research in Neuroscience). We appreciate Mr. Martin Sieber and Miss Huan-Ting Lu (Bionet Corp., Taipei, Taiwan) for the supply of SHEDS. The authors declare no conflicts of interest.

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