Aleš Hejčl

Bone Marrow Stem Cells and Polymer Hydrogels—Two Strategies for Spinal Cord Injury Repair

Summary1. Emerging clinical studies of treating brain and spinal cord injury (SCI) led us to examine the effect of autologous adult stem cell transplantation as well as the use of polymer scaffolds in spinal cord regeneration. We compared an intravenous injection of mesenchymal stem cells (MSCs) or the injection of a freshly prepared mononuclear fraction of bone marrow cells (BMCs) on the treatment of an acute or chronic balloon-induced spinal cord compression lesion in rats. Based on our experimental studies, autologous BMC implantation has been used in a Phase I/II clinical trial in patients (n=20) with a transversal spinal cord lesion.2. MSCs were isolated from rat bone marrow by their adherence to plastic, labeled with iron-oxide nanoparticles and expanded in vitro. Macroporous hydrogels based on derivatives of 2-hydroxyethyl methacrylate (HEMA) or 2-hydroxypropyl methacrylamide (HPMA) were prepared, then modified by their copolymerization with a hydrolytically degradable crosslinker, N,O-dimethacryloylhydroxylamine, or by different surface electric charges. Hydrogels or hydrogels seeded with MSCs were implanted into rats with hemisected spinal cords.3. Lesioned animals grafted with MSCs or BMCs had smaller lesions 35 days postgrafting and higher scores in BBB testing than did control animals and also showed a faster recovery of sensitivity in their hind limbs using the plantar test. The functional improvement was more pronounced in MSC-treated rats. In MR images, the lesion populated by grafted cells appeared as a dark hypointense area and was considerably smaller than in control animals. Morphometric measurements showed an increase in the volume of spared white matter in cell-treated animals. In the clinical trial, we compared intraarterial (via a. vertebralis, n=6) versus intravenous administration of BMCs (n=14) in a group of subacute (10–33 days post-SCI, n=8) and chronic patients (2–18 months, n=12). For patient follow-up we used MEP, SEP, MRI, and the ASIA score. Our clinical study revealed that the implantation of BMCs into patients is safe, as there were no complications following cell administration. Partial improvement in the ASIA score and partial recovery of MEP or SEP have been observed in all subacute patients who received cells via a. vertebralis (n=4) and in one out of four subacute patients who received cells intravenously. Improvement was also found in one chronic patient who received cells via a. vertebralis. A much larger population of patients is needed before any conclusions can be drawn. The implantation of hydrogels into hemisected rat spinal cords showed that cellular ingrowth was most pronounced in copolymers of HEMA with a positive surface electric charge. Although most of the cells had the morphological properties of connective tissue elements, we found NF-160-positive axons invading all the implanted hydrogels from both the proximal and distal stumps. The biodegradable hydrogels degraded from the border that was in direct contact with the spinal cord tissue. They were resorbed by macrophages and replaced by newly formed tissue containing connective tissue elements, blood vessels, GFAP-positive astrocytic processes, and NF-160-positive neurofilaments. Additionally, we implanted hydrogels seeded with nanoparticle-labeled MSCs into hemisected rat spinal cords. Hydrogels seeded with MSCs were visible on MR images as hypointense areas, and subsequent Prussian blue histological staining confirmed positively stained cells within the hydrogels.4. We conclude that treatment with different bone marrow cell populations had a positive effect on behavioral outcome and histopathological assessment after SCI in rats; this positive effect was most pronounced following MSC treatment. Our clinical study suggests a possible positive effect in patients with SCI. Bridging the lesion cavity can be an approach for further improving regeneration. Our preclinical studies showed that macroporous polymer hydrogels based on derivatives of HEMA or HPMA are suitable materials for bridging cavities after SCI; their chemical and physical properties can be modified to a specific use, and 3D implants seeded with different cell types may facilitate the ingrowth of axons.

HPMA-RGD Hydrogels Seeded with Mesenchymal Stem Cells Improve Functional Outcome in Chronic Spinal Cord Injury

Chronic spinal cord injury (SCI) is characterized by tissue loss and a stable functional deficit. While several experimental therapies have proven to be partly successful for the treatment of acute SCI, treatment of chronic SCI is still challenging. We studied whether we can bridge a chronic spinal cord lesion by implantation of our newly developed hydrogel based on 2-hydroxypropyl methacrylamide, either alone or seeded with mesenchymal stem cells (MSCs), and whether this treatment leads to functional improvement. A balloon-induced compression lesion was performed in adult 2-month-old male Wistar rats. Five weeks after injury, HPMA-RGD hydrogels [N-(2-hydroxypropyl)-methacrylamide with attached amino acid sequences--Arg-Gly-Asp] were implanted into the lesion, either with or without seeded MSCs. Animals with chronic SCI served as controls. The animals were behaviorally tested using the Basso–Beattie-Breshnahan (BBB) (motor) and plantar (sensory) tests once a week for 6 months. Behavioral analysis showed a statistically significant improvement in rats with combined treatment, hydrogel and MSCs, compared with the control group (P < 0.05). Although a tendency toward improvement was found in rats treated with hydrogel only, this was not significant. Subsequently, the animals were sacrificed 6 months after SCI, and the spinal cord lesions evaluated histologically. The combined therapy (hydrogel with MSCs) prevented tissue atrophy (P < 0.05), and the hydrogels were infiltrated with axons myelinated with Schwann cells. Blood vessels and astrocytes also grew inside the implant. MSCs were present in the hydrogels even 5 months after implantation. We conclude that 5 weeks after injury, HPMA-RGD hydrogels seeded with MSCs can successfully bridge a spinal cord cavity and provide a scaffold for tissue regeneration. This treatment leads to functional improvement even in chronic SCI.

Acute and delayed implantation of positively charged 2-hydroxyethyl methacrylate scaffolds in spinal cord injury in the rat

OBJECT Hydrogels are nontoxic, chemically inert synthetic polymers with a high water content and large surface area that provide mechanical support for cells and axons when implanted into spinal cord tissue. METHODS Macroporous hydrogels based on 2-hydroxyethyl methacrylate (HEMA) were prepared by radical copolymerization of monomers in the presence of fractionated NaCl particles. Male Wistar rats underwent complete spinal cord transection at the T-9 level. To bridge the lesion, positively charged HEMA hydrogels were implanted either immediately or 1 week after spinal cord transection; control animals were left untreated. Histological evaluation was performed 3 months after spinal cord transection to measure the volume of the pseudocyst cavities and the ingrowth of tissue elements into the hydrogels. RESULTS The hydrogel implants adhered well to the spinal cord tissue. Histological evaluation showed ingrowth of connective tissue elements, blood vessels, neurofilaments, and Schwann cells into the hydrogels. Morphometric analysis of lesions showed a statistically significant reduction in pseudocyst volume in the treated animals compared with controls and in the delayed treatment group compared with the immediate treatment group (p < 0.001 and p < 0.05, respectively). CONCLUSIONS Positively charged HEMA hydrogels can bridge a posttraumatic spinal cord cavity and provide a scaffold for the ingrowth of regenerating axons. The results indicate that delayed implantation can be more effective than immediate reconstructive surgery.

A new model of severe neurogenic pulmonary edema in spinal cord injured rat

We describe a new model of neurogenic pulmonary edema in spinal cord injured Wistar male rats. The pulmonary edema was elicited by an epidural thoracic balloon compression spinal cord lesion, performed under a low concentration of isoflurane (1.5 or 2%) in air. Anesthesia with 1.5% isoflurane promoted very severe interstitial and intraalveolar neurogenic pulmonary edema with a significantly increased thickness of the alveolar walls and massive pulmonary hemorrhage. In this group, 33% of animals died. Anesthesia with 2% isoflurane promoted severe interstitial and intraalveolar neurogenic pulmonary edema with less thickening of the alveolar walls and pulmonary hemorrhage. For evoking severe neurogenic pulmonary edema in spinal cord injured rats, 2% isoflurane anesthesia would be more suitable. However, if very severe neurogenic pulmonary edema needs to be evoked, spinal cord injury under 1.5% isoflurane anesthesia could be used, but one-third of the animals will be lost.

The use of diffusion tensor images of the corticospinal tract in intrinsic brain tumor surgery: a comparison with direct subcortical stimulation.

BACKGROUND Diffusion tensor imaging (DTI) is now widely used in neurosurgery to preoperatively delineate the course of the pyramidal tract. OBJECTIVE To evaluate the accuracy of the method by comparison with subcortical electrical stimulation and to evaluate the influence of the distance of the pyramidal tract from the tumor on the resection extent and postoperative clinical deficits. METHODS A diffusion tensor imaging depiction of the pyramidal tract was used in preoperative planning and intraoperative navigation in 72 cases. In 36 cases, subcortical electrical stimulation was used during the resection. The preoperative tumor-to-tract distance was compared with the stimulation result, the extent of resection, and the short-term postoperative course. RESULTS A significant nonlinear relationship between the tract-to-tumor distance and the probability of a motor response to subcortical stimulation was observed. The largest preoperatively measured tumor-to-tract distance with a positive stimulation result was 8 mm. Moreover, we observed a trend toward transient postoperative motor deterioration in patients with tumors close to the pyramidal tract. Resection extent was not significantly affected by the tumor-to-tract distance. CONCLUSION Despite methodological obstacles, reasonable accuracy of the diffusion tensor imaging reconstructions of the pyramidal tracts was confirmed by our study. The occurrence of transient postoperative motor deterioration is higher in patients with tumors located close to the pyramidal tract.

Adjusting the Chemical and Physical Properties of Hydrogels Leads to Improved Stem Cell Survival and Tissue Ingrowth in Spinal Cord Injury Reconstruction: A Comparative Study of Four Methacrylate Hydrogels

Currently, there is no effective strategy for the treatment of spinal cord injury (SCI). A suitable combination of modern hydrogel materials, modified to effectively bridge the lesion cavity, combined with appropriate stem cell therapy seems to be a promising approach to repair spinal cord damage. We demonstrate the synergic effect of porosity and surface modification of hydrogels on mesenchymal stem cell (MSC) adhesiveness in vitro and their in vivo survival in an experimental model of SCI. MSCs were seeded on four different hydrogels: hydroxypropylmethacrylate-RGD prepared by heterophase separation (HPMA-HS-RGD) and three other hydrogels polymerized in the presence of a solid porogen: HPMA-SP, HPMA-SP-RGD, and hydroxy ethyl methacrylate [2-(methacryloyloxy)ethyl] trimethylammonium chloride (HEMA-MOETACl). Their adhesion capability and cell survival were evaluated at 1, 7, and 14 days after the seeding of MSCs on the hydrogel scaffolds. The cell-polymer scaffolds were then implanted into hemisected rat spinal cord, and MSC survival in vivo and the ingrowth of endogenous tissue elements were evaluated 1 month after implantation. In vitro data demonstrated that HEMA-MOETACl and HPMA-SP-RGD hydrogels were superior in the number of cells attached. In vivo, the highest cell survival was found in the HEMA-MOETACl hydrogels; however, only a small ingrowth of blood vessels and axons was observed. Both HPMA-SP and HPMA-SP-RGD hydrogels showed better survival of MSCs compared with the HPMA-HS-RGD hydrogel. The RGD sequence attached to both types of HPMA hydrogels significantly influenced the number of blood vessels inside the implanted hydrogels. Further, the porous structure of HPMA-SP hydrogels promoted a statistically significant greater ingrowth of axons and less connective tissue elements into the implant. Our results demonstrate that the physical and chemical properties of the HPMA-SP-RGD hydrogel show the best combination for bridging a spinal cord lesion, while the HEMA-MOETACl hydrogel serves as the best carrier of MSCs.

Distant white-matter diffusion changes caused by tumor growth

OBJECTIVES Various reports have suggested that the involvement of normal-looking white matter with tumors is not limited to just signal abnormalities detectable on conventional imaging. Thus, the purpose of this study was to investigate the distant effects of glioblastomas and metastases on white matter using diffusion tensor imaging (DTI). MATERIALS AND METHODS Data for 21 patients harboring a glioblastoma (n=12) or a metastasis (n=9) located at a distance of smaller or equal to 10mm from a DTI-based reconstruction of the pyramidal tract were analyzed, using regions of interest (ROIs) placed along the pyramidal tracts in the cerebral peduncle distant (>15 mm) from the tumor. RESULTS For the whole study population, fractional anisotropy (FA) was significantly lower on the side ipsilateral to the tumor (P<0.001), a difference that was also observed in the glioblastoma and metastasis subgroups. The trace value was significantly higher on the ipsilateral side in the whole population and metastasis subgroup, but not in the glioblastoma subgroup. The decrease in FA and the trace value increase were significant in a subgroup of patients with motor deficits, but not in those without such deficits. CONCLUSION Hemispheric glioblastomas and metastases located close to the pyramidal tract induce diffusion changes in the tract that are observable at a distance of greater than 15 mm from the tumor border in the absence of T2 signal changes. These changes are different in glioblastomas and metastases, and mechanisms other than Wallerian degeneration may be contributing to the observed changes.

The role of nitric oxide in the development of neurogenic pulmonary edema in spinal cord-injured rats: the effect of preventive interventions.

Neurogenic pulmonary edema (NPE) is an acute life-threatening complication following an injury of the spinal cord or brain, which is associated with sympathetic hyperactivity. The role of nitric oxide (NO) in NPE development in rats subjected to balloon compression of the spinal cord has not yet been examined. We, therefore, pretreated Wistar rats with the NO synthase inhibitor N(G)-nitro-L-arginine methyl ester (L-NAME) either acutely (just before the injury) or chronically (for 4 wk prior to the injury). Acute (but not chronic) L-NAME administration enhanced NPE severity in rats anesthetized with 1.5% isoflurane, leading to the death of 83% of the animals within 10 min after injury. Pretreatment with either the ganglionic blocker pentolinium (to reduce blood pressure rise) or the muscarinic receptor blocker atropine (to lessen heart rate decrease) prevented or attenuated NPE development in these rats. We did not observe any therapeutic effects of atropine administered 2 min after spinal cord compression. Our data indicate that NPE development is dependent upon a marked decrease of heart rate under the conditions of high blood pressure elicited by the activation of the sympathetic nervous system. These hemodynamic alterations are especially pronounced in rats subjected to acute NO synthase inhibition. In conclusion, nitric oxide has a partial protective effect on NPE development because it attenuates sympathetic vasoconstriction and consequent baroreflex-induced bradycardia following spinal cord injury.

Identification of the large descending tracts using diffusion tensor imaging in Chiari III malformation

IntroductionThe paper focuses on the use of diffusion tensor imaging (DTI) in the evaluation of one case of Chiari III malformation.Case reportIn the case discussed, DTI was used to delineate the position of large descending tracts within the malformation and the reconstructed images were used to plan the surgical procedure.DiscussionThe clinical and imaging findings, the technical aspects of the DTI fiber tract reconstruction and the outcome are summarized.

Experimental reconstruction of the injured spinal cord

Injury to the spinal cord, with its pathological sequelae, results in a permanent neurological deficit. With currently available tools at hand, there is very little that clinicians can do to treat such a condition with the view of helping patients with spinal cord injury (SCI). On the other hand, in the last 20 years experimental research has brought new insights into the pathophysiology of spinal cord injury; we can divide the time course into 3 phases: primary injury (the time of traumatic impact and the period immediately afterwards), the secondary phase (cell death, inflammation, ischemia), and the chronic phase (scarring, demyelination, cyst formation). Increased knowledge about the pathophysiology of SCI can stimulate the development of new therapeutic modalities and approaches, which may be feasible in the future in clinical practice. Some of the most promising experimental therapies include: neurotrophic factors, enzymes and antibodies against inhibitory molecules (such as Nogo), activated macrophages, stem cells and bridging scaffolds. Their common goal is to reconstitute the damaged tissue in order to recover the lost function. In the current review, we focus on some of the recent developments in experimental SCI research.