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Intranasal Stem Cell Treatment as a Novel Therapy for Subarachnoid

Hemorrhage

Article  in  Stem Cells and Development · January 2018

DOI: 10.1089/scd.2017.0148

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ORIGINAL RESEARCH REPORTS

Intranasal Stem Cell Treatmentas a Novel Therapy for Subarachnoid Hemorrhage

Cora H. Nijboer,1,* Elke Kooijman,1,2,* Cindy T. van Velthoven,1,{ Erik van Tilborg,1 Ivo A. Tiebosch,3

Niels Eijkelkamp,1 Rick M. Dijkhuizen,3 Jozef Kesecioglu,2 and Cobi J. Heijnen4

Subarachnoid hemorrhage (SAH) represents a major health problem in Western society due to high mortalityand morbidity, and the relative young age of patients. Currently, efficacious therapeutic options are verylimited. Mesenchymal stem cell (MSC) administration has been shown to improve functional outcome andlesion size in experimental models of stroke and neonatal hypoxic-ischemic brain injury. Here, we studied thetherapeutic potential of intranasally administered bone marrow-derived MSCs relatively late postinsult using arat endovascular puncture model for SAH. Six days after induction of SAH, rats were treated with MSCs orvehicle through nasal administration. Intranasal MSC treatment significantly improved sensorimotor and me-chanosensory function at 21 days after SAH. Gray and white matter loss was significantly reduced by MSCtreatment and the number of NeuN+ neurons around the lesion increased due to MSC treatment. Moreover,intranasal MSC administration led to a sharp decrease in SAH-induced activation of astrocytes and microglia/macrophages in the lesioned hemisphere, especially of M2-like (CD206+) microglia/macrophages. Interest-ingly, MSC administration also decreased SAH-induced depression-like behavior in association with a resto-ration of tyrosine hydroxylase expression in the substantia nigra and striatum. We show here for the first timethat intranasal MSC administration reverses the devastating consequences of SAH, including regeneration ofthe cerebral lesion, functional recovery, and treatment of comorbid depression-like behavior.

Keywords: mesenchymal stem cells, intranasal treatment, subarachnoid hemorrhage, neuroregeneration,neuroinflammation, depression-like behavior

Introduction

The mortality rate of subarachnoid hemorrhage(SAH) in humans is approximately 40%–50% and sur-

viving patients are confronted with a great loss in quality oflife [1,2]. During SAH, blood flows into the subarachnoidspace of the brain, which induces several physiologicalchanges including an increase in cerebral pressure, a de-crease in oxygen tension, and an influx of peripheral im-mune cells, all of which contribute to cerebral brain damage.After the initial injury, which is caused by transient cerebralischemia and direct effects of presence of blood in thesubarachnoid space, secondary brain injury can develop dueto delayed cerebral ischemia (DCI). DCI is considered to bea major contributor to increased mortality and morbidityafter SAH and can be evoked through multiple pathophys-

iological mechanisms such as formation of cerebral edema,vasospasm, cortical spreading depolarization, and micro-thromboemboli [3–5].

The long-term outcome of SAH includes motor, speech,and cognitive dysfunction [6]. Furthermore, there is evi-dence for sensory symptoms in the subgroup of convexitySAH patients [7].

Moreover, depression is reported by one-third of thepatients even 12 months after the insult [8]. The limitedtreatment options available at present are mainly aimed atpreventing vasospasm to reduce DCI. However, the efficacyof these therapies is currently debated [5,9–11]. In view of thegreat need for novel efficacious therapies for SAH, also witha longer therapeutic time window, we studied the potential ofmesenchymal stem cells (MSCs) as a regenerative strategy inthe endovascular puncture rodent model of SAH.

1Laboratory of Neuroimmunology and Developmental Origins of Disease, Division Woman and Baby, University Medical CenterUtrecht, Utrecht, the Netherlands.

2Department of Intensive Care Medicine, University Medical Center Utrecht, Utrecht, the Netherlands.3Biomedical MR Imaging and Spectroscopy Group, Center for Image Sciences, University Medical Center Utrecht, Utrecht, the

Netherlands.4Laboratory of Neuroimmunology, Department of Symptom Research, Division of Internal Medicine, The University of Texas MD

Anderson Cancer Center, Houston, Texas.*Both these authors contributed equally to this work.{Current affiliation: Department of Neurology and Neurological Sciences, Stanford University School of Medicine, Stanford, California.

STEM CELLS AND DEVELOPMENT

Volume 27, Number 5, 2018

� Mary Ann Liebert, Inc.

DOI: 10.1089/scd.2017.0148

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Several studies using models of ischemic brain injury, suchas stroke and neonatal hypoxia–ischemia (HI), have shownthat MSC administration improves functional neurologicalrecovery and decreases brain lesion volume [12–16]. MSCtreatment up to even 10 days after HI in neonatal mice hassignificant beneficial effects on functional outcome and brainlesion size [12,17]. Furthermore, our recent data in the neo-natal HI model clearly indicate that transplanted MSCsstimulate endogenous repair by stimulation of the differ-entiation of neural progenitors into adult neurons [18–20].

In several experimental studies, MSCs have been adminis-tered either intracranially or systemically. Intravenous or intra-arterial injection of MSCs, however, results in a high accu-mulation of the cells in peripheral organs where they are notneeded [21]. Moreover, when administered systemically, ahigher number of MSCs is needed to obtain the same functionaleffect as we found with intranasal administration. For clinicalapplication, the intracranial route is too invasive [22].

To target the brain, stem cells have also been appliedthrough the nasal route [17,22–26]. We previously demon-strated that in neonatal mice with HI brain injury, intranasaltreatment with MSCs significantly decreased brain damageand improved functional outcome [17]. MSCs were capableof migrating selectively from the cribriform plate toward theipsilateral cerebral lesion site in 2–24 h. Furthermore, recentdata show that xenogeneic or allogeneic transplantation(with human or allogeneic murine MSCs, respectively) hassimilar regenerative capacity as mouse MSCs in a neonatalHI mouse model [22].

The goal of this study was to determine whether intra-nasal administration of MSCs at 6 days after experimentalSAH reduces cerebral lesion volume and neuroinflamma-tion, and improves functional outcome measured at 21 daysafter induction of SAH.

Materials and Methods

Animals and experimental procedure SAH

Experiments were performed in accordance with Dutchregulations, the European international guidelines (Directive86/609, ETS 123, Annex II), and approved by the ExperimentalAnimal Committee of the University Medical Center Utrecht(license Nos. 2012.I.04.057 and 2013.I.02.023). The experi-ments are reported in compliance with the Animal Research:Reporting of In Vivo Experiments (ARRIVE) guidelines.Adult male Wistar rats (Charles River, Maastricht, The Neth-erlands) of 300–350 g were housed in groups of four in Euro-standard Type IV cages (Tecniplast, Milan, Italy) with chipbedding and rat polycarbonate retreat (Bio-Serv/Plexx, Elst,The Netherlands) on reversed light/dark cycle (12 h light cycle)with ad libidum food access. Rats were anesthetized by me-chanical ventilation with 2% isoflurane in air/oxygen (2:1).An intramuscular injection of 5 mg/kg gentamicin (Cen-trafarm, Ettenleur, The Netherlands) was administered toprevent infections. Core temperature was maintained at37.5�C using a temperature-controlled heating pad. SAHwas induced as described previously [27,28]. In short, asharpened 3.0 Prolene suture was introduced into the rightinternal cerebral artery (ICA) up to the bifurcation of the ICAand the middle cerebral artery where the Circle of Willis wasperforated. Animals received 0.03 mg/kg buprenorphine(Reckitt & Colman, Kingston-Upon-Hill, UK) for pain relief.

Sham-operated rats underwent exactly the same procedure andanesthetic ventilation duration, without endovascular puncture.For magnetic resonance imaging (MRI) studies of the brainduring the acute phase after SAH in this model, we refer to ourearlier work [29,30]. Daily weight loss was monitored andweight loss >10 g per 24 h was compensated by subcutaneousinjection of 5 mL Ringer’s lactate (Baxter, Utrecht, TheNetherlands). Animals were randomly assigned to four differ-ent experimental groups: SAH animals treated with either ve-hicle n = 10 or MSCs n = 13, and sham-operated rats treatedwith either vehicle n = 13 or MSCs n = 9. Group sizes weredetermined based on variability in our previous experimentsusing the same techniques [27]. We could not include someof the rat brains because the quality of the slices in the regionof interest was not sufficient. The decision to exclude thosesamples was made before unblinding the groups.

MSC administration

Bone marrow-derived MSCs of Sprague Dawley rats werepurchased from Invitrogen [GIBCO Rat (SD) MSCs; LifeTechnologies] and cultured according to the manufacturer’sinstructions. At 6 days post-SAH, MSCs or vehicle [phosphatebuffered saline (PBS)] was delivered intranasally in awakeanimals. Thirty minutes before MSC or vehicle adminis-tration, two doses of 6mL hyaluronidase in PBS (total 100 U;Sigma-Aldrich, Steinheim, Germany) were applied to eachnostril and spontaneously inhaled [17,24]. Hyaluronidase de-grades hyaluronic acid, thereby increasing tissue permeability,which is necessary for administration of larger particles likecells. Subsequently, a total of 1.5 · 106 MSCs in 24mL PBS or24mL vehicle solution was administered in two doses of 6mLapplied to each nostril. The method of intranasal delivery ofcells has been extensively described by Danielyan et al. [23].

Functional outcome

All behavioral experiments were performed in a blindedmanner.

Neurological score

Acute neurological deficits to define the severity of theinsult were measured before SAH (day 0) and at days 1--7post-SAH by an adaptation of the method described by Su-gawara et al. [31] and described earlier by our group [27].The scoring includes measurement of spontaneous activity,spontaneous movements of all limbs, outstretching while heldby tail, climbing wall of wire cage, and response to vibrissaetouch. The maximum score was 15 in absence of any deficits.

We recently showed that there is a wide variety in neu-rological score and hemorrhage size in the endovascularpuncture model due to the unpredictable degree of bleeding[27]. We showed that SAH animals with a neurological score>12 (‘‘mild SAH’’) at day 1 do not develop a clear cerebrallesion nor a deficit in sensorimotor function [27]. To be ableto monitor changes in cerebral lesion volume and functionaloutcome, we, therefore, selected the group of animals withsevere neurological disabilities of £12 (‘‘severe SAH’’).

Sensorimotor function

Sensorimotor function was tested by the adhesive re-moval task (ART) at 21 days post-SAH. Adhesive labels

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(Tough-Spots; Diversified Biotech, Boston, MA) were placedon the left and right forepaw, and the latency to removal wasrecorded. The mean time until complete removal of threestickers per forepaw was recorded. Sticker placement onleft and right forepaws was alternated between and withinanimals. The ART was performed in the dark by a trainedobserver blinded to treatment.

Mechanosensory function

Mechanosensory function was measured at 21 days post-SAH using von Frey hairs [32]. Rats were placed in indi-vidual plastic enclosures on a mesh floor and allowed toacclimate for 30 min. A series of calibrated von Frey fila-ments (bending force range from 1 to 15 g; Stoelting, WoodDale, IL) was applied perpendicularly to the plantar surfaceof the hindpaw with sufficient force to bend the filaments for6 s, and brisk paw withdrawals, shaking, licking, or flinchingwere considered as a positive response. In the absence of aresponse, the filament of next greater force was applied. Ifa response occurred, the filament of next lower force wasapplied. The force in grams producing a 50% likelihood ofwithdrawal (50% withdrawal threshold) was calculated us-ing the up-and-down method as described [33].

Depression-like behavior

To determine depression-like behavior, we used the su-crose preference test [34]. At 21 days post-SAH, animalswere solitary housed with access to food, normal tap water,and 1% sucrose water. During the dark period, water and 1%sucrose were weighed every 4 h. The first day was used foracclimation of the animals and these results were not used fordata analysis. During the next 2 days, water and 1% sucrosewater intake were measured every 4 h. To determine whetherchanges in sucrose intake respond to the antidepressant effectof ketamine, we administered ketamine (Narketan 10; Veto-quinol S.A., Lure Cedex, France) intraperitoneally after thelast measurement on day 3 at a dose of 10 mg/kg [35]. Afteradministration of ketamine, sucrose preference was measuredfor 1 more day (on day 4). The sucrose preference test wasperformed in the dark by an observer blinded to the treatment.

Histology

Rats were euthanized at 21 days post-SAH by pentobarbitaloverdose and perfused with PBS followed by 4% parafor-maldehyde. Brains were postfixed, embedded in paraffin, andcoronal sections of 8 mm were cut at bregma. Deparaffin-ized sections were incubated with mouse-antimicrotubule-associated protein 2 (MAP2; 1:1,000; Sigma-Aldrich),mouse-antimyelin basic protein (MBP; 1:1,600; SternbergerMonoclonals, Lutherville, MD), or rabbit antityrosine hy-droxylase (TH; 1:1,000; Abcam, Cambridge, UK) followed bybiotin-labeled horse-antimouse or goat-antirabbit antibodies(Vector Laboratories, Burlingame, CA). Staining was revealedusing Vectastain ABC kit (Vector Laboratories) and diami-nobenzamidine with an addition of ammonium nickel sulfatefor the TH staining. The MAP2 and MBP data were analyzedby determining the ipsi-/contralateral positive area · 100%using Adobe Photoshop CS6 and ImageJ software (http://imagej.nih.gov/ij/index.html), respectively. TH-positive pixelswere analyzed using ImageJ software (http://imagej.nih.gov/ij/

index.html) with threshold and histogram plugins. By usingthis software, background staining was subtracted. The samethreshold was used for all animals. For the substantia nigra,we captured images of three microscopic fields on one slidefor each animal in the same area of the substantia nigra, bothipsilateral and contralateral, for all slides and quantified thepercentage area staining positively for TH. We did not ob-serve overt tissue damage in this area (no loss of MAP2staining, no cysts, and no apparent gliosis). In addition, wecaptured images of the entire striatum in each hemisphereon one slide per animal and assessed the number of positivepixels normalized for striatal area. In the striatum, we didobserve clear lesions in at least some of the areas and,therefore, we normalized for total striatal area.

For immunofluorescent stainings, sections were incu-bated with mouse-anti-NeuN (1:100; Chemicon), mouse-antiglial fibrillary acidic protein (GFAP; 1:200; CymbusBiotechnology, Southampton, UK), rabbit-anti-ionized cal-cium binding adaptor molecule 1 (Iba-1; 1:500; Wako),rabbit-anti-CD86 (1:200; AbD Serotec, Oxford, UK), orgoat-anti-CD206 (1:200; R&D Systems, Minneapolis, MN),followed by incubation with secondary antibodies AlexaFluor-488 goat-antimouse IgG, AlexaFluor-594 goat-antirabbit IgG,AlexaFluor-488 donkey-antigoat IgG, or AlexaFluor-488goat-antirabbit IgG (all Molecular Probes, Eugene, OR).Sections were counterstained with DAPI and were exam-ined with an Axio Observer Microscope with AxiovisionRel 4.6 software (Carl Zeiss, Sliedrecht, The Netherlands).For quantifying the number NeuN+ cells surrounding the le-sion site, NeuN+ cells were manually counted in two micro-scopic fields surrounding the lesion site or, if no lesion wasobserved, at similar locations in the cortex. Iba-1 and GFAPwere analyzed by determining the percentage Iba-1- orGFAP-positive expression per whole hemisphere per animal.CD86 and CD206 expression levels were assessed by mea-suring the number of CD86- or CD206- and DAPI-positivecells per mm2. CD86 and CD206 cell counts were assessed inthree microscopic fields in intact cortical areas surroundingthe infarct area both ipsilateral and contralateral so sixmicroscopic fields per animal. For all analyses, software ofImageJ was used (http://imagej.nih.gov/ij/index.html). Allanalyses were performed in a blinded manner.

Statistical analysis

Data were analyzed by one- or two-way analysis of vari-ance with Bonferroni post hoc tests. Data are presented asmean – standard error of the mean. Gray and white matterdamage (MAP2, MBP stainings), sensorimotor behavior, andneuroinflammation (Iba-1, GFAP stainings) were measuredas primary outcome parameters. Other markers were assessedas secondary outcome parameters.

Results

Acute neurological score after SAH

A total of 105 rats were subjected to the endovascularpuncture procedure (or sham operation) and severity of theinsult was determined by scoring neurological deficits fromdays 1 to 7 after the surgical procedure using a neurologicalscoring system of 1–15. A score of 15 represents ‘‘no def-icits’’ [27,31]. At day 6 after surgery, the neurological score

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of all surviving SAH animals had returned to levels of sham-control animals (Fig. 1A). In this study, only SAH animalswith a neurological score of £12 at day 1 (in total n = 46),which were still alive on day 6 (n = 23), were used, as we haveshown before that these animals (designated as ‘‘severe SAH’’)develop a cerebral lesion and functional deficits [27]. Themortality rate after severe SAH was 50% (23 out of 46 animalsin this group survived) and all mortality occurred withinthe first 6 days, before we started the MSC administration(Fig. 1B). The neurological scores at day 1 of SAH animalsthat received MSC administration (n = 13) were similar tothose of animals that received vehicle treatment (n = 10). Noneof the sham-operated animals (n = 22) died (Fig. 1B). ‘‘SevereSAH’’ rats showed significant weight loss in comparison with‘‘mild SAH’’ (a total of n = 37 that were not followed-up) andsham-control rats at 1–6 days post-SAH (Fig. 1C).

Intranasal treatment with MSCsand sensorimotor behavior

Animals with ‘‘severe SAH’’ and sham-control animalswere treated intranasally with 1.5 · 106 MSCs (sham-control: n = 9; SAH: n = 13) or vehicle (sham: n = 13; SAH:n = 10). To investigate whether treatment with MSCs im-proved sensorimotor function, we subjected all rats to theART at 21 days post-SAH. During the ART, the time ismeasured that the animal needs to remove a tiny adhesivepatch from its forepaw (‘‘total removal time’’). The ‘‘totalremoval time’’ can be split in the latency to start removal ofthe adhesive patch (‘‘latency to start removal’’) as a measureof sensory function, and the time it takes the animal toremove the patch after sensing it (‘‘effective adhesive re-moval time’’) as a measure of fine motoric function [36].

Vehicle-treated SAH animals (n = 10) showed a signifi-cant increase in ‘‘total removal time’’ of the adhesive patchfrom their left (impaired) forepaw compared with their right(unimpaired) forepaw or compared with vehicle-treatedsham-control rats (n = 13; Fig. 2A). In sham-control rats, the‘‘total removal time’’ of the adhesive patch between left andright forepaw did not differ (Fig. 2A). In addition, ‘‘latencyto start removal’’ and ‘‘effective adhesive removal time’’were both increased in the left (impaired) forepaw invehicle-treated SAH rats (Fig. 2B, C).

MSC treatment at 6 days post-SAH significantly de-creased ‘‘total removal time’’ of the left (impaired) forepaw(n = 13; Fig. 2A), ‘‘latency to start removal’’ (Fig. 2B), and‘‘effective adhesive removal time’’ (Fig. 2C) compared withvehicle treatment. Sham-operated animals treated withMSCs (n = 9) showed similar removal times in the ART asvehicle-treated sham-operated animals (Fig. 2A–C).

In the same group of rats, we measured mechanosensoryfunction after SAH by testing the sensitivity to touch of thehind paws using von Frey hairs [32,33]. This test uses nylonfilaments of various strengths to apply different but accurateforces to the hind paw to determine the mechanical thresholdto touch. The sensitivity to touch was significantly decreasedin vehicle-treated SAH animals compared with sham-operated animals at 21 days after SAH. Administration ofMSCs at 6 days after SAH significantly increased thesensitivity to touch represented by a decrease in the 50%threshold, indicating an improved mechanosensory re-sponse after MSC treatment in SAH animals (Fig. 2D).

Brain lesion size after SAH and MSC treatment

To determine whether treatment with MSCs at 6 daysafter SAH improves structural brain damage, we assessedgray and white matter loss by MAP2 and MBP staining,respectively, at 21 days after injury.

After SAH, rats showed an extensive brain lesion re-presented by a large decrease in ipsilateral MAP2 expression,

FIG. 1. Neurological scores, survival, and weight loss afterSAH. (A) Acute neurological scores from days 1 to 7 afterSAH are displayed for all rats that survived for 21 days [n = 22sham (green circles), n = 23 mild SAH (black squares), n = 23severe SAH (red triangles)]. Stratification in mild or severeSAH was based on the neurological score at day 1 usingneurological score >12 as ‘‘mild SAH’’ and neurological score£12 as ‘‘severe SAH.’’ (B) Survival curve from days 1 to 21after induction of SAH. Survival rate is shown for all animalsused in this study. Sham n = 22 (green), mild SAH n = 37(black), severe SAH n = 46 (red). (C) Weight loss from days 1to 6 post-SAH compared with weight before SAH at day 0.Sham n = 22 (green circles), mild SAH n = 23 (black squares),severe SAH n = 23 (red triangles). *P < 0.05, **P < 0.01,***P < 0.001 versus sham. Data represent mean – SEM. SAH,subarachnoid hemorrhage; SEM, standard error of the mean.Color images available online at www.liebertpub.com/scd

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mainly in the cortex (sham-control vehicle: n = 13; sham-control MSC: n = 9; SAH vehicle: n = 8; SAH MSC: n = 11;Fig. 3A, B). Treatment with MSCs at 6 days after SAH re-sulted in significantly less ipsilateral MAP2 loss comparedwith vehicle treatment at 21 days after SAH (Fig. 3A, B).MSC treatment in sham-operated animals did not have anyeffect on MAP2 expression compared with vehicle treatmentin sham-operated animals (Fig. 3B). To assess neuronal re-population around the SAH-induced lesion, the density ofNeuN+ cells in the lesion area was quantified. Figure 3C andD shows that the number of NeuN+ neurons surrounding thelesion site was reduced in vehicle-treated SAH animals incomparison with sham animals. Importantly, MSC treatmentsignificantly increased the number of NeuN+ cells in the areasurrounding the lesion site (sham-control vehicle: n = 6; SAHvehicle: n = 5; SAH MSC: n = 7; Fig. 3C, D).

SAH also induced white matter loss, represented by asignificant decrease in MBP staining in the ipsilateral cortex(Fig. 3E, F). MSC treatment reduced ipsilateral white matterloss as shown by an increased ratio of ipsilateral/contralat-eral MBP expression compared with vehicle treatment afterSAH (sham-control vehicle: n = 13; sham-control MSC:n = 9; SAH vehicle: n = 8; SAH MSC: n = 11; Fig. 3E, F).MSC treatment in sham-operated animals did not affectMBP expression (Fig. 3F).

Because we did not observe any effects of MSC treatmentin sham-control animals on behavioral outcome and/or grayand white matter damage, we did not include this experi-mental group in the further analyses.

Cerebral inflammation and MSC treatment

We recently showed that SAH induces a neuroinflammatoryresponse in the brain in rats that persists until at least 21 dayspost-SAH [27]. To evaluate the effect of MSC treatment onthis inflammatory response, expression of GFAP in the ipsi-lateral hemisphere as a marker for astrocyte activation wasdetermined. At 21 days post-SAH, GFAP expression was in-creased in vehicle-treated SAH animals compared with sham-operated animals, particularly at the border of the lesion(sham-control vehicle: n = 6; SAH vehicle: n = 6; SAH MSC:n = 8; Fig. 4A, B). MSC treatment after SAH significantlyreduced GFAP expression at the lesion border compared withvehicle treatment (Fig. 4A, B).

Macrophage/microglia activation, measured by Iba-1expression in the ipsilateral hemisphere, was 2.2-fold in-creased in vehicle-treated SAH rats compared with brainsof sham-operated animals (Fig. 4A). Increased Iba-1 ex-pression after SAH was mainly observed in the cortex andstriatum (Fig. 4A). Furthermore, the morphology of Iba-1-expressing macrophages/microglia in the ipsilateral cor-tex and striatum changed from small, finely branched,quiescent microglia observed in sham-operated animalstoward denser, less-branched, amoeboid-like activatedmicroglia in vehicle-treated SAH animals (Fig. 4A, in-sets). MSC treatment after SAH dramatically decreasedipsilateral Iba-1 expression compared with vehicle treat-ment (Fig. 4A, C). Moreover, after MSC treatment, themorphology of the Iba-1-positive cells appeared as ‘‘resting’’

FIG. 2. Long-term sensorimotorand mechanosensory behavior def-icits after SAH are attenuated afterMSC treatment. (A–C) Sensor-imotor function was tested usingthe ART at 21 days post-SAH. (A)The total removal time for both leftand right forepaws is presented inseconds for all experimentalgroups. (B) The latency to startremoval is presented in seconds.(C) The effective adhesive removaltime is presented in seconds. (D)Sensitivity to mechanical stimula-tion was determined using the vonFrey test at 21 days post-SAH.Data are expressed as 50% with-drawal threshold in grams.**P < 0.01 ***P < 0.001 versussham; ###P < 0.001 SAH+vehicleversus SAH+MSC. Data representmean – SEM. Sham n = 13, sham+MSC n = 9, SAH+vehicle n = 10,SAH+MSC n = 13. ART, adhesiveremoval task; MSC, mesenchymalstem cell.

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macrophages/microglia with typical ramified appearance(Fig. 4A, insets).

Next we evaluated microglia polarization toward a moreproinflammatory M1-like state or an anti-inflammatory/regenerative M2-like state. We determined CD86 expressionas a proinflammatory M1-like phenotype and CD206 expres-sion as a M2-like phenotype. CD206-positive macrophages/microglia have been shown to exert a debris-scavenging,

prohealing function [37]. Compared with sham-control rats,the number of CD86-positive cells was not altered afterSAH or after MSC treatment (sham-control vehicle: n = 10;SAH vehicle: n = 8; SAH MSC: n = 12; Fig. 5). In contrast,the number of CD206-positive cells was highly increased at21 days after SAH (Fig. 5B, D). Interestingly, MSC treat-ment reduced the number of CD206-positive cells to shamlevel (Fig. 5B, D).

FIG. 3. MSC treatment reduces gray and white matter loss and increases the number of neuronal cells populating thelesion site after SAH. (A) Effect of MSC treatment on gray matter damage at 21 days after SAH determined by neuronal-specific MAP2 staining. Photographs show representable pictures of MAP2 staining in sham-operated (top), vehicle-treated SAH (middle), and MSC-treated SAH animals (bottom). (B) Bar graph showing ipsi-/contralateral MAP2-positivearea · 100%. (C) Effect of MSCs on number of NeuN+ neurons (green) surrounding the lesion site (L) at 21 days post-SAH. Nuclei are stained with DAPI (blue). Scale bar: 100 mm. (D) The SAH-induced decrease in density of NeuN+ cellssurrounding the lesion is partly restored after MSC treatment. (E) Effect of MSC treatment on white matter damageat 21 days after SAH determined by myelin-specific MBP staining. Photographs show representable pictures ofMBP staining in sham-operated (top), vehicle-treated SAH (middle), and MSC-treated SAH animals (bottom). (F) Bargraph showing ipsi-/contralateral MBP-positive area · 100%. ***P < 0.001, ****P < 0.0001 sham versus SAH+vehicle,#P < 0.05 and ##P < 0.01 SAH+vehicle versus SAH+MSC. (A, B, E, F) Sham+vehicle n = 13; sham+MSC n = 9;SAH+vehicle n = 8; SAH+MSC n = 11. (C, D) Sham+vehicle n = 6; SAH+vehicle n = 5; SAH+MSC n = 7. Data representmean – SEM. MAP2, microtubule-associated protein 2; MBP, myelin basic protein. Color images available online atwww.liebertpub.com/scd

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The effect of MSC treatment on SAH-induceddepression-like behavior

One of the long-term consequences of SAH in patients isdevelopment of major depression with a prevalence of 33% at

12 months post-SAH [8]. To determine whether depression-like behavior is present in rats after SAH, we performed thesucrose preference test at 21 days post-SAH. This test has beenextensively used as a measure of depression-like behavior inrodents and captures lack of motivation and anhedonia [38,39].

FIG. 4. Long-term macrophage/mi-croglia and astrocyte activation afterSAH is reduced after MSC administra-tion. (A) Immunofluorescent photo-graphs showing macrophage/microgliaand astrocyte activation by Iba-1 stain-ing (red) and GFAP staining (green)respectively, in the ipsilateral hemi-sphere of five representative sham-operated, vehicle-treated SAH andMSC-treated SAH animals. Photo-graphs are taken at 21 days post-SAH.Insets show a higher magnification;scale bar in inset represents 300mm. (B)Effect of MSC treatment on GFAP ex-pression at 21 days after SAH. Bargraph shows percentage of GFAP ex-pression in the ipsilateral hemisphere.(C) Effect of MSC treatment on Iba-1expression at 21 days after SAH. Bargraph shows percentage of Iba-1 expres-sion in the ipsilateral hemisphere. Shamn = 6, SAH+vehicle n = 6, SAH+MSCn = 8. *P < 0.05, ***P < 0.001 sham ver-sus SAH+vehicle; #P < 0.05 SAH+vehicleversus SAH+MSC. Data representmean – SEM. GFAP, glial fibrillary acidicprotein; Iba-1, ionized calcium bindingadaptor molecule 1. Color images avail-able online at www.liebertpub.com/scd

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Sweetening of water by adding sucrose elicits a natural pref-erence over normal tap water in healthy rodents. Anhedoniahas been shown to reduce the preference for sucrose water[40]. In our study, sham-operated animals showed a su-crose preference of 88%, whereas vehicle-treated SAH ani-mals showed no preference for sucrose, indicating anhedonia(sham-control vehicle: n = 13; sham-control MSC: n = 9; SAHvehicle: n = 10; SAH MSC: n = 13; Fig. 6).

To further define whether anhedonia represents depression-like behavior, rats were treated with the pharmacologicalantidepressant, ketamine [41,42]. Ketamine has recently beendescribed to have an acute antidepressive effect in rodents aswell as in humans when given in a low dose [43]. Figure 6shows that a single dose of ketamine significantly reversedsucrose preference in vehicle-treated SAH animals.

Importantly, MSC treatment at 6 days after SAH com-pletely restored the sucrose preference of SAH animals to

the level of sham-operated animals. MSC treatment did notaffect sucrose preference in sham-operated animals (Fig. 6).The total fluid intake did not differ between all treatmentgroups (data not shown).

Influence of SAH and role of MSCson the dopaminergic system

Major depression in humans is often associated with awidespread loss of dopaminergic tonus and decreased do-pamine synthesis [38]. Moreover, dopaminergic neurons arevery sensitive to cerebral injury signals. TH converts tyro-sine to l-DOPA, a precursor of dopamine, and a decrease inTH staining can be considered as a decrease in dopamineproduction.

At 21 days post-SAH, TH staining was decreased in thenerve endings in the striatum (Fig. 7A, B) and in the substantia

FIG. 5. Effect of MSC treatment on polarization of microglia. (A) Representative immunofluorescent photographs of CD86staining (green) in contra- and ipsilateral hemispheres of sham-operated, vehicle-treated SAH, and MSC-treated SAH animals.Nuclear staining (DAPI) is depicted in blue. (B) Bar graph showing number of CD86-positive cells per mm2 in contra- andipsilateral hemispheres of all experimental groups. (C) Representative immunofluorescent photographs of CD206 staining(green) in contra- and ipsilateral hemispheres of sham-operated, vehicle-treated SAH, and MSC-treated SAH animals. Nuclearstaining (DAPI) is depicted in blue. (A, C) Photographs are taken at 21 days post-SAH. Scale bar represents 40mm. (D) Bargraph showing number of CD206-positive cells per mm2 in contra- and ipsilateral hemispheres of all experimental groups.***P < 0.001 sham-ipsi versus SAH+vehicle-ipsi and #P < 0.05 SAH+vehicle-ipsi versus SAH+MSC-ipsi. Data are presented asmean – SEM. Sham n = 10; SAH+vehicle n = 8; SAH+MSC n = 12. Color images available online at www.liebertpub.com/scd

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nigra (Fig. 7C, D) of the ipsilateral hemisphere of vehicle-treated SAH rats compared with sham animals (sham-controlvehicle: n = 11; SAH vehicle: n = 9; SAH MSC: n = 12). THstaining in the contralateral substantia nigra in SAH animalswas indistinguishable of that in sham animals (Fig. 7C, D).MSC treatment increased ipsilateral TH staining in cell bodiesof the substantia nigra and nerve endings in the striatum tolevels in sham animals (Fig. 7A–D).

Discussion

We demonstrated for the first time that intranasal ad-ministration of MSCs at 6 days after severe SAH improvessensorimotor function and decreases lesion size in rats.Moreover, neuroinflammation associated with SAH, as-sessed by astrocyte and microglia/macrophage activation,was significantly reduced by MSC treatment [27]. Finally,intranasal administration of MSCs reversed the depression-like state of rats induced by SAH, which was associatedwith a restoration of TH expression in the ipsilateral sub-stantia nigra and the striatum.

The advantage of using MSCs over current pharmacolog-ical strategies to combat the negative consequences of SAH isthe capacity of MSCs to repair already damaged tissue as wealso have shown for MSC treatment in our mouse model ofhypoxic-ischemic brain injury [17,22]. In that model, the le-sion size was reduced by approximately 50%–60% in re-sponse to intranasal administration of MSCs. Khalili et al.showed a beneficial effect of intravenous MSC transplanta-tion in the single endogenous blood injection SAH model[44,45]. MSCs were administered at 24 h after SAH in thismodel and showed an improvement in neurological scorefrom 7.75 to 3.5 at 14 days post-SAH. A comparison betweenthis study and the study performed by Khalili et al. is diffi-cult since different readouts were used and different animal

models of SAH were applied using different administrationroutes of the MSCs [44]. Instead of intravenous administra-tion, we used a noninvasive and easy route of intranasal ad-ministration. Although we did not assess infiltration of theMSCs into the brain in this study, recent studies by Donegaet al. and van Velthoven et al. showed in a neonatal mousemodel of HI that nasal administration of MSCs allows mi-gration of the MSCs specifically to the damaged brain areas[17,22,24], probably through a chemokine-mediated pathway.Interestingly in this model of unilateral brain damage, thenasally administered MSCs accumulate predominantly in thehemisphere in which the damage was present, even thoughthey were administered through both nostrils [17,24]. Theeffective intranasal delivery of stem cells to the rodent brainhas also been shown in several other adult models of neu-rodegeneration such as Parkinson’s disease, stroke, multiplesclerosis, Alzheimer’s disease, and brain tumors and is nowwidely used and accepted [23,25,26,46–48]. Recent unpub-lished data showed that besides the migration toward thedamaged brain area in small rodents, intranasal administeredMSCs are also capable of migrating to the lesioned area in anewborn baboon subjected to carotid occlusion (article inpreparation). These data indicate that even when the migra-tion route is considerably longer in the newborn monkey, theMSCs still arrive at the damaged brain area. The latter dataare of translational importance when this intranasal route willbe used for patients with SAH.

We started our MSC treatment relatively late, that is, at6 days after the insult, to allow for full establishment of thelesion, including damage caused by possible delayed cere-bral ischemia (DCI) which have an onset at days 1–3 afterthe endovascular puncture. Previous MRI studies by us andothers in this SAH model have shown that increased tissueperfusion, leakage of blood–brain barrier, edema formation,and further tissue degeneration, including gliosis and ne-crosis, lead to a well-established lesion at day 7 after SAH[29,30,49]. We, therefore, suggest that the beneficial effectsof MSC transplantation at day 6 after SAH are the result ofregenerative repair mechanisms activated by MSCs. This isin line with our earlier findings showing that MSC trans-plantation alters the gene expression profile and induces newneuronal precursor formation in the subventricular zone(SVZ) in a model of neonatal brain damage [19,50]. Wepropose that activation of endogenous repair mechanismsunderlies the beneficial effects of MSC treatment. Indeedour present data indicate that MSC treatment induces anincrease in NeuN+ cells surrounding the lesion area whencompared with vehicle treatment in the current SAH model(Fig. 3). Even though it remains to be determined what theexact mechanism is that underlies the increase in NeuN+ cells,our data point toward promotion of repair rather than a pro-tective effect of MSCs. This is clinically relevant because aregenerative mechanism allows for an extended therapeuticwindow, which is urgently needed for SAH patients.

We observed unilateral brain damage after SAH in thehemisphere ipsilateral to the endovascular puncture, whichis in line with other studies using the endovascular puncturemodel. Only in cases of very severe bleeding, some studieshave shown involvement of the contralateral hemisphere[27,51]. Delayed ischemia, possibly resulting from vaso-spasms, is an important secondary process that might con-tribute to unilateral brain damage after experimental SAH

FIG. 6. MSC administration attenuates SAH-induceddepression-like behavior. Depression-like (anhedonic) be-havior was tested using the sucrose preference test. Theintake of normal tap water and sucrose water was measuredevery 4 h and preference for the sucrose water (%) wascalculated. *P < 0.05, **P < 0.01, ***P < 0.001 versus nopreference (50%) (dotted line). Data are presented as mean –SEM. Sham+vehicle n = 13, sham+MSC n = 9, SAH+vehiclen = 10, SAH+MSC n = 13, SAH+ketamine n = 10.

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[52–54]. Vasospasms are most prominent near the site of theaneurysm [3], this would be a possible explanation for theunilateral brain damage that is observed.

One of the striking consequences of SAH is persistentabundant microglia activation in the damaged hemisphereas represented by an increase in Iba-1 staining that waseven present at 21 days after SAH [27]. However, althoughthe microglia showed a morphology associated with anactivated state, we only observed an increase in CD206-positive staining of microglia after SAH and not of theCD86-positive phenotype. CD206-expressing microglia/macrophages have been associated with tissue regenerationand repair [37]. CD206-positive cells typically phagocytosered blood cells or tissue debris, recruit anti-inflammatoryT cells, and cause a downregulation of proinflammatorycytokine production [37]. Moreover, CD206 itself is involved

in the binding of apoptotic and necrotic cells [55,56].Therefore, we suggest that the overwhelming presence ofCD206-positive microglia in the brains of SAH animals isprobably a late response to the presence of erythrocytes,apoptotic cells, and tissue debris.

Jose et al. showed that MSCs reduce microglia prolifer-ation in vitro [57], which is in accordance with the decreasein Iba-1 expression after MSC administration post-SAHas we show in Fig. 4. In this respect, it is of interest thatMSC treatment prevented the expression of CD206-positivemicroglia/macrophages, whereas the expression of CD86-positive microglia/macrophages remained at a similar levelas after vehicle treatment. This apparent contradiction of areduced number of regenerative microglia/macrophages af-ter MSC treatment may be explained by the fact that MSCswhen given at day 6 reduce the proinflammatory state of the

FIG. 7. MSC administration increases TH expression after SAH. Effect of MSC administration on dopaminergic tonus at21 days after SAH determined by TH expression in striatum (A, B) and substantia nigra (C, D). (A) Representativephotographs of TH staining at the contralateral (contra) and ipsilateral (ipsi) striatal level of sham-operated, vehicle-treatedSAH, and MSC-treated SAH animals. Scale bar represents 50mm. (B) Bar graph showing effect of MSC treatment on THexpression in the contra- and ipsilateral hemisphere at striatal level. (C) Representative photographs of TH staining in thecontralateral (contra) and ipsilateral (ipsi) substantia nigra of sham-operated, vehicle-treated SAH, and MSC-treated SAHanimals. Scale bar represents 50 mm. (D) Bar graph showing effect of MSC treatment on TH expression in the contra- andipsilateral hemisphere in the substantia nigra as quantified in the area indicated by the square in the overview images.##P < 0.01 SAH+vehicle-contra and -ipsi versus SAH+MSC-contra and -ipsi, *P < 0.05 SAH+vehicle-ipsi versus sham-ipsiand versus SAH+MSC-ipsi. Data represent mean – SEM. Sham n = 11, SAH+vehicle n = 9, SAH+MSC n = 12. TH, tyrosinehydroxylase.

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brain, which prevents further deterioration of brain damageand thereby abolishes the need for a late polarization ofmicroglia to the M2-like state.

The beneficial effects of treatment with MSCs also in-clude an improvement of sensorimotor function. The in-creased time to sense the presence of the patch in the ARTin SAH animals indicates a loss of sensory function. Thisconclusion is further strengthened by our data in the vonFrey test that showed higher threshold to sense light sensorystimuli applied to the paws of SAH rats.

We observed that brain damage after SAH predominantlyoccurs at the cortical level and spreads, depending on theintensity of insult, from layer I in the S1/M1 cortex to layerVI [27]. The clear reduction in cortical damage after MSCtreatment may possibly explain the improvement in senso-rimotor behavior after MSC transplantation.

In humans, depression is a frequent symptom after SAH. Ina prospective study, Kreiter et al. showed that 38% of SAHpatients have depressive symptoms at 3 months after the in-sult and 33% of patients are still depressed at 12 monthspostinsult [8]. Moreover, up to 50% of SAH patients en-counter cognitive impairments, which is thought to contributeto poor recovery and reduced quality of life after SAH. Areduction in depressive symptoms would most likely improvethe quality of life and overall recovery of SAH patients.

In the rodent endovascular puncture model of SAH, weobserved depression-like behavior at 21 days post-SAH.Although this might be a relative early time point after SAH,rats lost their preference for sucrose water, and this anhe-donic state has been defined as depression-like behavior inmultiple animal studies [58,59]. Structural brain damageevoked by SAH may cause a loss of sucrose preference by adamage-induced loss of taste and/or olfactory function.However, a single acute treatment with a low dose of ke-tamine as an antidepressant drug reversed SAH-inducedanhedonia, indicating that the depression-like behavior isnot the result of loss of taste/olfactory function.

The mechanisms underlying the depression-like state af-ter SAH and the beneficial effects of MSC treatment remainunclear however. We investigated changes in the dopami-nergic system and show a unilateral decrease in TH stainingin the substantia nigra and a decrease in TH in nerve endingsin the striatum after SAH. These findings indicate a reduc-tion in dopamine production that may contribute to the an-hedonia as observed in the sucrose preference test. MSCtreatment restored TH staining and normalized sucrosepreference, supporting the notion that loss of dopamineoutput and impaired sucrose preference may be related.

Early release of proinflammatory cytokines possibly asa response to the acute insult may induce depression-like behavior by upregulation of the enzyme indoleamine2,3-dioxygenase, leading to an increase in quinolinic acid, a neu-rotoxic NMDA receptor agonist known to induce depression-like behavior [43,60,61].

A decrease in BDNF expression has also been proposedas a mechanism for establishing a depressed state in animalsand humans [62]. In the double-hemorrhage model of SAH,Jiang et al. showed an increased ratio between pro-BDNFand mature BDNF expression in the brain, indicating a de-creased expression of mature BDNF [63]. Our group hasshown previously that MSCs increase their BDNF produc-tion when encountering an ischemic brain milieu [19,48].

MSCs likely also increase BDNF levels in the brain dam-aged by SAH and this is another potential mechanism forsuppression of the observed anhedonia.

In conclusion, we demonstrate for the first time that asingle intranasal delivery of MSCs has the potential to be-come an effective therapeutic strategy with a longer thera-peutic time window than currently available therapies tocombat the devastating effects of SAH. Moreover, intranasalMSC therapy does not only repair the brain structure andimprove sensorimotor function after the insult, but also re-verses the depression-like state induced by SAH.

Acknowledgments

This study was funded, in part, by the ‘‘Dirkzwager-Assink Foundation’’ and by the ‘‘Friends of UMC Utrechtfund.’’ The authors thank S. Versteeg for excellent technicalassistance with the von Frey test.

Author Disclosure Statement

No competing financial interests exist for any of the authors.

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Address correspondence to:Dr. Cobi J. Heijnen

Laboratory of NeuroimmunologyDivision of Internal Medicine

Department of Symptom ResearchThe University of Texas MD Anderson Cancer Center

Unit 14501515 Holcombe Boulevard

Houston, TX 77030

E-mail: cjheijnen@mdanderson.org

Received for publication July 18, 2017Accepted after revision January 8, 2018

Prepublished on Liebert Instant Online January 8, 2018

INTRANASAL MSC THERAPY FOR SUBARACHNOID HEMORRHAGE 325

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