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Available online at www.sciencedirect.com

Neuroscience Letters 430 (2008) 169–174

Neurotransplantation in mice: The concorde-like position ensuresminimal cell leakage and widespread distribution of cells

transplanted into the cisterna magna

Miroslaw Janowski a,b, Magdalena Kuzma-Kozakiewicz c, Donat Binder d, Hans-Jorg Habisch d,Aleksandra Habich a, Barbara Lukomska a, Krystyna Domanska-Janik a,

Albert C. Ludolph d, Alexander Storch e,∗a Department of Neurorepair, Medical Research Center, Warsaw, Poland

b Department of Neurosurgery, Medical University of Warsaw, Polandc Department of Neurology, Medical University of Warsaw, Poland

d Department of Neurology, University of Ulm, Germanye Department of Neurology and Center for Regenerative Therapies Dresden, Technical University of Dresden,

Fetscherstrasse 74, 01397 Dresden, Germany

Received 5 September 2007; received in revised form 15 October 2007; accepted 30 October 2007

bstract

The access of transplanted cells to large areas of the CNS is of critical value for cell therapy of chronic diseases associated with widespreadeurodegeneration. Intrathecal cell application can match this requirement. Here we describe an efficient method for cell injection into the cisternaagna and the assessment of the cell distribution within subarachnoidal space in mice. In order to maximize cell distribution we applied a “concord-

ike” position, where the cisterna magna is nearly the highest point of the animal’s body. A drop of saline on the needle insertion site avoided theutflow of transplanted cells from subarachnoidal space with CSF during surgery. Twenty-four hours later the preparation of the CNS with an intactura mater by a suitable dissection technique (described in detail) revealed approx. 80% of the injected cells (100,000 cells per animal) within the

ubarachnoidal space ranging from the skull base (olfactory nerve to premedullary cisterns) to the IV ventricle, and to both the ventral and dorsalurfaces of the spinal cord. Thus the “concorde-like” position proved to be very useful for intrathecal cell application leading to a widespread cellistribution within the subarachnoidal space. 2007 Elsevier Ireland Ltd. All rights reserved.

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eywords: Neurotransplantation; Stem cell therapy; Cisterna magna; Concorde

ell replacement therapy for diseases other than hematopoietictem cell substitution is still in the preclinical stage. For neuro-ransplantation a number of various stem cell types are currentlyeing investigated [5,9,12], including embryonic or neural stemells [13], bone marrow stem cells (MSCs) [4,6] and umbilicalord blood cells [1,2,8]. Methods of cell transplantation varyrom most invasive (intracerebral, intraspinal) to minimal inva-

ive (intrathecal, systemic). Although being minimal invasive,ntrathecal application via lumbar puncture seems to be inferioro other methods, because the injection takes place at the very

∗ Corresponding author. Tel.: +49 351 458 2532; fax: +49 351 458 4352.E-mail address: alexander.storch@neuro.med.tu-dresden.de (A. Storch).

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304-3940/$ – see front matter © 2007 Elsevier Ireland Ltd. All rights reserved.oi:10.1016/j.neulet.2007.10.050

position; Dura preparation

downstream” end of the CSF circulation, probably prohibitingell delivery to more rostral regions. Additionally, this methods technically difficult in experimental rodents. Relatively largeells like those used in the present study, i.e. human bonearrow-derived mesenchymal stem cells (hMSCs), demand for

sing needles with a diameter wide enough to permit cell pen-tration. Injections into the cisterna magna were successfullysed by others in rat models of rapid-onset CNS disorders, suchs traumatic brain injury [7], spinal cord injury [10,15], andtroke [16]. In case of chronic neurodegenerative diseases with

disseminated neuronal loss in various areas of the CNS, such

s amyotrophic lateral sclerosis (ALS) or Alzheimer’s diseaseAD), cell replacement strategies demand a widespread accessf the transplanted cells to large areas of the CNS, and likely

170 M. Janowski et al. / Neuroscience Letters 430 (2008) 169–174

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ig. 1. Photographs of the mouse head fixed in the stereotactic frame accordingfter placing a drop of saline solution on top of the surgical field in order to avof the whole CNS with intact dura mater as described in detail in the text (C–E)

eed repetitive injections (implying the use of minimal invasiveethods). One major drawback of cisterna magna injections is

igh cell leakage which could not be controlled in previous stud-es, and subsequently the authors shifted to intraventricular cellpplication [10].

Here we describe a technique of intracisternal cell applica-ion in mice, which enabled us to avoid significant cell leakagend to achieve a widespread distribution of transplanted cellsithin the subarachnoidal space. Human MSCs were isolated

s previously described [4]. Prior to transplantation, cells wereabeled with 1 �g/ml Hoechst 33342 (Sigma, St. Louis, MO) and�g/ml 5-(and-6)-carboxyfluorescein diacetate, succinimidylster (CFSE; Molecular Probes, Eugene, OR) for 30 min at7 ◦C, washed twice with PBS and further incubated with freshedium for 24 h in order to minimize efflux of non-binding

ye. Prior to transplantation the cells were suspended in PBSt a density of 10,000 �l−1. B6SJL mice were purchased fromackson Laboratories (Ben Harbor, ME, USA) and maintainedn the animal facility of the University of Ulm. All experimen-al procedures have been approved by the Care Committee ofxperimental Animals in Tubingen, Germany (registration no.16).

Before surgery, anesthesia was induced by atropine followedy ketamine (100 mg/kg) and xylazine (16 mg/kg). A stereo-axic apparatus (ASI Instruments, Heidelberg, Germany) wasmployed to fix the head of the animal. The posterior part of the

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e “concorde-like position” after insertion of the transplantation needle (A) andoutflow of the cell suspension through the needle tract (B). (C–G) PreparationCNS shown from above (F) and below (G).

pparatus was lifted to form a 30◦ angle with the table surface.fter fixing the head in ear bars, we put a roller under the mouse.he size of the roller was adjusted so that the line connecting

he most prominent parts of the cranium and the spine formed a5◦ angle with the horizontal line (Fig. 1A). The tooth bar wassed to press the head down on the nasion, so that the line deter-ining the facial surface formed a 15◦ angle with the vertical

ine. The lines together formed a 90◦ angle (Fig. 1A). In thatosition, the cisterna magna was nearly at the highest point ofhe mouse body.

For application of cells, we made a midline skin incision fromhe superior nuchal line to the level of C3 vertebrae under theperating microscope, followed by a strict midline blunt dissec-ion of muscles. As the atlanto-occipital membrane was exposed,0 �l of cells (10,000 �l−1) were taken into a Hamilton syringeHamilton, 20 �l). The syringe was then placed in the pumpWPI, Ultra Micro Pump), which was mounted on a microma-ipulator arm. Subsequently, the needle (gauge 30, Hamilton,oint Style 4) was positioned over the midline of the atlanto-ccipital membrane (where its anterior–posterior dimension ishe longest) to form an angle of 60◦ with the horizontal line.he latter line corresponds to the line directly leading towards

he cerebello-medullary fissure, which is visible under the oper-ting microscope through the membrane. The membrane wasouched by the needle tip between the occiput and the pos-erior arch of atlas. It was important that the membrane was

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M. Janowski et al. / Neuros

trained to facilitate its penetration with the needle, which coulde achieved by slightly varying the inclination of the animal’sead. The membrane was quickly pierced and the needle wastopped exactly in half of the needle tip slope. It allowed theutflow of CSF through the needle tip providing space for celluspensions to be injected without excessive increase of CSFressure. The needle was then moved forward to position thehole needle tip slope inside the cisterna magna. Under the oper-

ting microscope, it was possible to control the needle endingnside the cisterna magna to protect the brain stem from injury.ext, a large drop of saline (approximately 100 �l) was placedn top of the surgical field (Fig. 1B). Cells were injected overperiod of 10 min. During the injection it was indispensable tobserve the animal’s breathing movements, and, as in one casef relenting and subsequent stopping of breathing, the needlead to be partially withdrawn until the animal had recovered.ollowing injection of the cells the needle was kept in place for0 more minutes. After needle withdrawal, 10 �l of the salinelaced on top of the surgical field were collected and revealedhe presence of some erythrocytes and leukocytes, but only a fewransplanted Hoechst 33342- and CFSE-labeled cells (no morehan 100 cells per 10 �l aliquot (1000 transplanted cells or 1%n toto), reflecting minimal cell leakage. Finally, the wound waslosed with staples (3 M, Neuss, Germany).

Twenty-four hours after transplantation mice were perfusedith saline followed by 2% paraformaldehyde (PFA) [11] and

he whole bodies were postfixed in 2% PFA overnight. This fix-tion procedure proofed to be best suited for the subsequentreparation, because it made the tissue stable enough—withhe appropriate softness, and still enabled immunohistochem-cal staining. The dissection of the whole CNS with intact dura

ater was the most critical factor for adequate evaluation of cellistribution within the subarachnoidal space. Thus we workedut an appropriate method of dissection. Initially, limbs, thorax,nd abdomen were cut off the trunk of the body. Soft tissuesike muscles, tendons, and fat were dissected off the skeletono expose the skull and the vertebral column. Further dissectionas performed under the microscope.The ligamentum flavum stretching between the vertebral

rches in the lumbar region was cut with tiny scissors in order tonter the vertebral canal and to expose the cauda equina. Then,he rostral arch was cut and the vertebral canal was opened byreaking off both vertebral pedicles with forceps and taking offhe vertebral arch including the spinous process exposing thepinal cord with its whitely lucent dura mater. Subsequently, allertebral arches were removed using this technique (Fig. 1C)ntil the C2 level was reached. The vertebral bodies thoughere left intact in order to stabilize the spinal cord. Then, theosterior part of C1 was taken off with special attention in ordero expose, not to tear the atlanto-occipital membrane with theubjacent cisterna magna. Since the atlanto-occipital membranend the cranial parts of the dura tightly adhere to occipital bone,heir separation required special attention. The bone was bro-

en into little bits, which could then be carefully detached fromhe dura. Then, the skull base was prepared. The cranial nerveseaving the brain and entering the skull base bone foramina asell as the pyramid of the temporal bone additionally complicate

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e Letters 430 (2008) 169–174 171

he procedure. Importantly, one had to avoid any level motiono prevent the bones from drilling into the brain parenchyma.fter the removal of the lateral, frontal, and parietal bones, theasal crest, which till then had served as anchorage point for thengers to lock the CNS into position (Fig. 1D), was removed.inally, the vertebral bodies were taken off (Fig. 1E). Using thisrocedure we were able to preserve the dura mater around thehole CNS (Fig. 1F and G). Eventually, the CNS was cryopre-

erved in 30% sucrose for 24 h, frozen in isopentane at −56 ◦C,nd stored at −80 ◦C.

Prior to histological analyses of cell distribution, the dis-ected CNS was cut on a cryostat (Leica CM3050 S) at 40 �mhick slices. The slices were evaluated for the presence of there-labeled cells under an Axiovert 135 inverse fluorescenceicroscope (Carl Zeiss, Gottingen, Germany). Estimation of

ell numbers within the subdural space was performed by count-ng Hoechst-labeled cell nuclei on every third slice throughouthe CNS. The precise counting was sometimes restrained byight clusters of cells found within the subdural space prohibit-ng single cell detection. Where feasible, single cell nuclei wereounted, else estimates based on the area occupied by the celllusters compared to the area of single cells were evaluated.

The cell density of every cluster was comparable andccounted for approximately 5000–10,000 cells per cluster. Theighest number of cells was found in the cerebello-pontinengle and the ambient and basal cisterns ranging from 15,000 to0,000 cells distributed over both sides of each mentioned space.pproximately 10,000 cells were found around the spinal cord

90% in the cervical and 10% in the thoracic region), and an aver-ge of 5000 cells in the IV ventricle, the prepontine, premedullar,nd large cisterns respectively, as well as on both sides of theptic chiasm and the olfactory nerve cisterns (Fig. 2). In both theuadrigeminal cistern and the III ventricle the total number ofells did not exceed 1000. No cells were found in the lateral ven-ricles and on brain convexity. In total, we found approximately0% of transplanted cells in all compartments described above.he variation between individual mice was minimal (n = 5).

The herein established transplantation method was subse-uently employed for the transplantation of more than 150ransgenic ALS mice with various stem cell populations observ-ng no surgery-related complications applying this technique [3].our mice died during the first night after surgery. This occurrednly once during the transplantation period, on 2 consecutiveays. No other animals died prematurely following surgery,ence we consider the applied surgical method as being safe.

The only route to bypass the blood–brain barrier in miceeaving the nervous tissue intact is the penetration of the cis-erna magna. We first started transplantations using the techniqueescribed for the collection of CSF [14] which resulted in aignificant cell leakage also described by other authors [10].fter needle withdrawal we observed a rapid efflux of CSF con-

aining transplanted cells due to the CSF pressure, significantlynhanced by gravity. The cell leakage was in part a consequence

f the animal’s position during the procedure, where the cisternaagna was close to the lowest point of the cranium. This position

an be successfully used for CSF collection, but in order to min-mize the influence of gravitation, we applied the widely used

172 M. Janowski et al. / Neuroscience Letters 430 (2008) 169–174

Fig. 2. Representative microphotographs of different sites of the subarachnoidal space of brain (A, olfactory nerve cistern; B, optic chiasm cistern; C, basal cistern; D,ambient cistern E, prepontine cistern; F, cerebello-pontine cistern; G, IV ventricle) and spinal cord (H, ventral surface of cervical spinal cord) showing the distributionof hMSCs 24 h after transplantation into the cisterna magna using the “concorde-like position” method. Arrows indicate the transplanted cells stained with Hoechst33342 (blue) and CFSE (green). Scale bar: 200 �m. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of thearticle.)

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ule of neurosurgery in humans that the surgical field shoulde the highest point of the cranium and translated the “con-orde position” from human neurosurgery to our animal settingith the cisterna magna being the highest point of the mouseody.

The other important factor contributing to cell leakage afterhe adjustment of the mouse position was the CSF pressure. Inhe concorde-like position the operative wound was lying in theorizontal plain enabling us to place a drop of saline within theound during the injection procedure (Fig. 1B). By force ofravity, the saline significantly enhanced protection against celleakage. The diffusion between CSF and saline was irrelevant, asnly single cells were found in saline after the procedure (∼1%).hus, 24 h after surgery cell recovery within the subarach-oidal space reached approximately 80% of the transplantedells.

In contrast to other forms of intrathecal or intraparenchymalell transplantation, the use of the concorde-like position causesell sedimentation from the injection site towards both the cra-ial and caudal direction. A significant number of cells coulde retrieved at the skull base and in the IV ventricle (which areocated against the CSF stream) indicating that the cell distribu-ion depends not only on gravitation, but also on the pressure ofhe administrated fluid. The CSF circulation seems to be insuf-cient to disperse the large cell clusters during the first day after

ransplantation.There are only a few descriptions of the fate of cells following

n injection into the cisterna magna. They are all based on ratodels and rat-derived cells. A Chicago group employed MR

maging to follow the transplanted cells in a model of stroke16]. After transplantation, the cells were found only in theisterna magna. During the following week nearly the wholeell population revealed a pilgrimage towards the stroke area.imilarly, a Chinese group revealed the presence of rat bonearrow cells injected into the cisterna magna exclusively at the

ite of the lesion in a model of traumatic brain injury [7]. Aapanese group applied hippocampus-derived neurospheres inspinal cord injury model. The transplanted cells were foundithin the subarachnoidal space of the spinal cord 1 week

fter injection [15]. Almost no cells were visualized in theII and the lateral ventricles. The authors did not report theresence of cells at the skull base, but these cells could haveeen missed during the brain dissection without the dura. Inur case we developed a method for the preparation of thehole CNS with an intact dura mater, and were thus able

o investigate the cell distribution within the entire subduralpace. Since we could not find a description of this techniquefter extensive literature search, we delineate it in detail in thistudy.

Although the final cell distribution might depend on theroperties of the transplanted cell type and the type of brainissue lesion, the application of the “concord-like position”or cell injection into the cisterna magna, as introduced in

he present study, uniformly distributes the transplanted cellsithin the subarachnoidal space of the brain and the spinal

ord. Particularly for chronic neurodegenerative diseases withdisseminated pattern of cell loss like ALS or AD, we

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e Letters 430 (2008) 169–174 173

ecommend the “concorde-like position” for intrathecal cellransplantation.

cknowledgements

We appreciate the excellent technical assistance of Steffeneier and Nancy Meyer. This study was supported by grants

rom the Ministry of Scientific Research and Information Tech-ology of Poland grant no. K053/P05/2003 (MJ, MK, AH, BL),he Bundesministerium fur Bildung und Forschung (Federal

inistry of Science and Technology), Polish-German Coopera-ion in Neuroscience Program (grant no. 01GZ0313; ACL, AS),nd the Landesstiftung Baden-Wurttemberg (ForderprogrammAdulte Stammzellen’ grant no. 37610; AS).

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