The Intrathecal Infusion of Mesenchymal Stem Cells into Healthy Rabbits is Safe and Devoid of Neurological or Clinical Complications

Background: Cellular, molecular and transplantation studies using mouse and rat models of neurological diseases have revealed that Mesenchymal Stem Cells (MSC) represent a reliable candidate for cellular therapies for neurological conditions, including Amyotrophic Lateral Sclerosis (ALS) and Spinal Cord Injury (SCI). However, evaluation of safety aspects associated with route and dose of cell infusion, pivotal issues in cell therapy, has not been tested in large animals.


Introduction
A growing interest in the use of adult stem cells for the treatment of several neurological disorders has been developed over the last few years. This is the result of a better understanding of stem cell properties as well as encouraging conclusions from studies in neurologicaldamaged animals after treatment with stem cells [1][2][3].
The appealing molecular and cellular properties of Mesenchymal Stem Cells (MSC), a type of adult stem cells [4,5], have prompted the concept that these cells represent a reliable candidate for cell therapy for neurological diseases [6][7][8]. The above assertion is supported by three major facts: a) MSC can differentiate outside the mesenchymal lineage into neurological precursors [5,9,10], b) after infusion of MSC to animals with genetic or experimental neurological diseases, MSC migrate and survive within the central nervous system [6,11] and contribute to the improvement/recovery of pre-symptomatic motor functions [6,7,[11][12][13] and c) MSC secrete several growth factors and chemokines that may create a neuroprotective environment favorable for tissue sparing and axonal regeneration [14,15 ].
A salient issue in cell therapy is the choice of a proper and safe route of administration of the cell product. Numerous studies have been performed to evaluate neuroplasticity, but not safety, after infusion (intravenous, intrathecal, subarachnoidal space or cortex) of MSC to mice models of motor neuron diseases [13,[16][17][18][19]. In turn, intrathecal infusion [12,20] emerges as an attractive option for cell delivery to treat neurological patients. However, there are no feasible data to assert that the intrathecal infusion of MSC to humans is safe and devoid of any serious neurotoxicity.
This study was performed to evaluate, prior the initiation of a clinical trial using MSC [21], whether the intrathecal infusion of MSC to healthy rabbits is feasible, safe and devoid of neurological damage. As compared to mice, rabbits which are phylogenetically, anatomical and physiological closer to humans, are also large enough for monitoring and evaluating physiological changes after an injury.

Animals
Male New Zealand white normal rabbits (12 weeks, 2.7-3.2 kg body weight, Harlan Laboratories, www.harlan.com) were used for bone marrow aspiration and for intrathecal MSC infusion. Rabbits were kept in cages and received food and water without restrictions. All animal studies were done in accordance with the National Institutes of Health Guidelines for the care and use of mammals in Neuroscience and Behavioral Research (http://grants.nih.gov/grants/olaw/National_ Academies_Guidelines_for_Use_and_Care.pdf).
After aspiration, animals were taken to their cages and had free access to water and food. Rectal temperature was monitored during the next 2 days. Three days after bone marrow aspiration, animals were sacrificed by a lethal injection of pentobarbital (200 mg/kg).

MSC expansion
For MSC isolation and expansion, described procedures [22,23] were followed with minor modifications. Briefly, bone marrow aspirates were diluted with Dulbecco's Phosphate Buffered Saline (DPBS, Sigma Aldrich, St Louis, Mo, USA), and processed by gradient density separation (Histopaque-1077; Sigma Aldrich, St Louis, Mo, USA). The resulting fraction of mononuclear cells was suspended in α-MEM (Invitrogen, Carlsbad, CA, USA) containing 10 % fetal bovine serum (Hyclone, Logan, UT, USA), seeded in 25 cc culture flasks (1x10 6 MNC/cm 2 ) and incubated (5% CO 2 , 37°C) for 9 days. Cells in the developing adherent layer (MSC) were expanded by 3 successive passages. Culture medium was changed every 3 days. At the end of the expansion process, resulting MSC cells were prepared for cryopreservation and storage.
The day before cell infusion, frozen MSC were thawed, resuspended carefully in culture medium and subjected to a short incubation period (24 hrs, 37°C, 5% CO 2 ) to remove cell aggregates and excess of DMSO. Resulting cells were resuspended in infusion medium (IM, DPBS containing 0.05 % donor rabbit serum) and filtered (BD Falcon Cell Strainer, Nylon Mesh 100-µm). Aliquots of the cell product were taken to assess cell number, viability (Trypan blue exclusion), endotoxin content (Limulus Amebocyte Lysate assay), microbiological condition (Fluid Thioglycollate Medium and Gram's stain) and for immunophenotype characterization [23].

Cell infusion
For intrathecal infusion, proper aliquots of the cell product were taken and resuspended in IM. A low (0.3 x 10 6 MSC/kg) and a high cell dose (0.6 x 10 6 MSC/kg) product was prepared and transferred to 1 cc syringes. When translated to a clinical setting, the above doses will be comparable to the infusion of 0.1 x 10 6 /kg to 2.5 x 10 6 MSC /kg, which is similar to the range of MSC utilized or proposed in clinical studies [21,[24][25][26].
For infusion, animals were anesthetized (IV sodium pentobarbital, 25 mg/kg), placed on the operating surface with back in a flexed position and the intervertebral space (between L6 and L7) prepared. A spinal needle (25 GA, 2.00 in; 0.50 x 51 mm) was inserted into the canal space and the precise position of the catheter was confirmed by cautious aspiration of a small volume (0.2 cc) of cerebrospinal fluid. The cell products or the placebo solution (IM, no cells) were injected into the spinal fluid during a period of 1 minute. After removing the needle, the animals were placed head up for 30 minutes and then returned to their cages.

Neurological and behavioral follow-up
In anticipation that MSC infusion may trigger focal or diffuse changes (cortex, subcortex and/or spinal cord), animals were routinely inspected (days 3, 7, 14, 21 and 34) for the onset of distinctive signals , which in the rabbit have been associated with the development of Nystagmus (non-controlled eye movement and/or asymmetry of pupils) [27,28], Myoclonous (involuntary twitching of one or a group of muscles) [29] and/or Hydrocephalous (eyes with 'setting-sun' appearance and/or abnormal sounds after fingertips on the skull) [30].
In turn, behavioral evaluations were done including changes in feeding (eating, drinking or coprophagy) and spontaneous (conduct inside the cage) manners [31]. Scores assigned to individual items were adjusted to yield a final score of 3 and 4 points for normal feeding (FB) and spontaneous (SB) behavior, respectively. Any increase over these scores was considered as abnormal.

Histological studies
After sacrifice, the whole spinal cord and brain of each animal were dissected and grossly examined for evidence of morphologic abnormalities. The tissue was placed in 10 percent neutral buffered formalin and fixed for greater than 48 hours. Serial sections of forebrain, midbrain, hindbrain, brainstem, cerebellum and spinal cord (cervical, thoracic and lumbar) were cut, processed and embedded in paraffin. Sections were cut (at 3-4 µ), stained with hematoxylin-eosin and examined by a pathologist who was unaware of the animal group and of possible neurologic outcomes. The sections were examined for evidence of necrosis, inflammation, neoplasia and architectural distortion and atrophy. Since, there is abundant information describing MSC survival and migration within the CNS [10,11], sections were not elsewise examined for engraftment of MSC.

Clinical assessment after infusion
Rectal temperature in all animals was found to be within normal ranges for healthy rabbits (38.3°C -39.4°C) during the entire evaluation period. Similarly, hematological values (CBC) were within normal ranges, as reported for rabbits kept under laboratory conditions [32].
Throughout the time course of this study (34 days) and irrespective of the type of infusion received (low or high cell dose or placebo), body weight increased in all animals. The average increase in body weight during the study period was 25%, which is within the reported frequency distribution of weight for young New Zealand white rabbits [33].

Neurologic assessment after infusion
After intrathecal infusion, animals were subjected to neurological evaluation at baseline and once a week. Results in Table 1 and 2 shows that during the entire period of evaluation and irrespective of the type of infusion received, clinical signs for nystagmus, myoclonus or hydrocephalus were not detected.

Feeding and spontaneous behavior assessment
The procedures associated with the administration of the cell or placebo product, brought forth changes (day +3) in the Feeding (FB) and Spontaneous (SB) behavior scores of all animals. These abnormal scores demonstrated to be transient in nature, since by day +7 normal behavior scores were detected in nearly all animals (Table 1 and 2).

Histological assessment
Serial sections of forebrain, midbrain, hindbrain, cerebellum and of each level of spinal cord of each animal were examined and showed no evidence of inflammation, necrosis, atrophy or neoplasia. There was no evidence of any architectural distortion. Representative photographs of sections are shown in (Figure 2).

Discussion
Despite there is an abundance of information concerning the infusion of mesenchymal stem cells to small animals (murine models of neurological diseases), scarce information is available about the safety and feasibility of the infusion of MSC to large animals. Accordingly, this study was designed to assess whether the intrathecal infusion of MSC into healthy rabbits was procedurally practicable, safe and devoid of side effects. Intrathecal infusion by lumbar puncture is considered as a unique technique for minimally invasive cell transplantation [12]. In addition, the infused cells mediated by cerebrospinal fluid circulation [11], are transported to the damaged tissue in the spinal cord and/or brain.
The results obtained demonstrated that: a) the procedure for the   For abbreviations, see Table 1   Table 2: Long-term evaluation of clinical and neurological symptoms after intrathecal or placebo infusion of MSC.
intrathecal infusion by lumbar puncture of MSC was uncomplicated.
After the forward movement of the spinal needle into the spinal canal (L7) and aspiration of a small volume of CSF, the cell product was delivered into the spinal fluid in less than 2 minutes, b) after infusion, changes in the Feeding (FB) and Spontaneous (SB) behavior of the animals were detected; however effects were transient and vanished in almost all animals by day 7 after infusion. Since these transitory modifications in animal demeanor were equally detected in cell-and placebo-infused animals, we speculate that their origin was mainly associated with lumbar puncture maneuvers rather than to infusion of cells into the spinal canal, c) additional strength to the notion that the infusion of MSC by lumbar puncture is not harmful, was given by the observation that the increase in body weight during the evaluation period was similar in control and cell-infused animals. This observation is important since preclinical studies have shown that delayed body weight development is secondary to vascular or neurological damage [34], d) no signs of neuropathological findings (suggestive of brain lesions, focal CNS damage and/or obstruction of CSF flow) [35], were detected during the follow up period ( Table 2, N/M/H column) both in treated and control animals, and finally, e) the histopathological examination confirmed in the tissue specimens prepared from all animals, normal histology and no evidence of increased inflammation or cellular atypia. Thus, our study demonstrated that the intrathecal infusion of MSC can be readily performed under safe and feasible conditions without inducing neurological and histological changes.
As mentioned in the Introduction, a main issue in cell therapy is to identify not only a proper and safe route for cell infusion, but the assurance that security persists with time and is not dependent on the number of cells infused.
As this was be case in this study, the translation of these findings to a clinical setting put forward the acquiescence that nearly nine months after MSC infusion, patient's safety will persists. Alike time is consider long enough to prove security in a clinical setting [2].
Consequently, our results showing that intrathecal infusion of MSC is safe and feasible, in addition to data demonstrating MSC survival/ migration within the CNS [6,13] and effectiveness in neurological recovery [6,15], epitomize a weighty foundation for their use in the treatment of neurological patients.