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Reverse thermal gels have numerous biomedical implications, as they undergo physical gelation upon temperature increases and can incorporate biomolecules to promote tissue repair. Such a material is developed for the sustained release of bevacizumab (Avastin), a drug used to treat age-related macular degeneration. The polymer, poly(ethylene glycol)-poly-(serinol hexamethylene urethane) (ESHU), forms a physical gel when heated to 37 °C and shows good cytocompatibility with ocular cells. ESHU is capable of sustaining bevacizumab release over 17 weeks in vitro, and the release kinetics can be altered by changing the drug dose and the ESHU concentration. These results suggest that ESHU is biologically safe, and suitable for ocular drug delivery.

In amyotrophic lateral sclerosis (ALS), the exogenous temporal triggers that result in initial motor neuron death are not understood. Overactivation and consequent accelerated loss of vulnerable motor neurons is one theory of disease initiation. The vulnerability of motor neurons in response to chronic peripheral nerve hyperstimulation was tested in the SOD1(G93A) rat model of ALS. A novel in vivo technique for peripheral phrenic nerve stimulation was developed via intra-diaphragm muscle electrode implantation at the phrenic motor endpoint. Chronic bilateral phrenic nerve hyperstimulation in SOD1(G93A) rats accelerated disease progression, including shortened lifespan, hastened motor neuron loss and increased denervation at diaphragm neuromuscular junctions. Hyperstimulation also resulted in focal decline in adjacent forelimb function. These results show that peripheral phrenic nerve hyperstimulation accelerates cell death of vulnerable spinal motor neurons, modifies both temporal and anatomical onset of disease, and leads to involvement of disease in adjacent anatomical regions in this ALS model.

Astrocytes play important roles in normal CNS function; however, following traumatic injury or during neurodegeneration, astrocytes undergo changes in morphology, gene expression and cellular function known as reactive astrogliosis, a process that may also include cell proliferation. At present, the role of astrocyte proliferation is not understood in disease etiology of neurodegenerative diseases such as amyotrophic lateral sclerosis (ALS), a fatal motor neuron disorder that is characterized by a relatively rapid degeneration of upper and lower motor neurons. Therefore, the role of astrocyte proliferation was assessed in both acute and chronic mouse models of motor neuron degeneration, neuroadapted sindbis virus (NSV)-infected mice and SOD1(G93A) mice, respectively. While astrocytes proliferated in the lumbar spinal cord ventral horn of both disease models, they represented only a small percentage of the dividing population in the SOD1(G93A) spinal cord. Furthermore, selective ablation of proliferating GFAP(+) astrocytes in 1) NSV-infected transgenic mice in which herpes simplex virus - thymidine kinase is expressed in GFAP(+) cells (GFAP-TK) and in 2) SOD1(G93A) × GFAP-TK mice did not affect any measures of disease outcome such as animal survival, disease onset, disease duration, hindlimb motor function or motor neuron loss. Ablation of dividing astrocytes also did not alter overall astrogliosis in either model. This was likely due to the finding that proliferation of NG2(+) glial progenitors were unaffected. These findings demonstrate that while normal astrocyte function is an important factor in the etiology of motor neuron diseases such as ALS, astrocyte proliferation itself does not play a significant role.

The efficacy of protein-based therapies for treating injured nervous tissue is limited by the short half-life of free proteins in the body. Affinity-based biomaterial delivery systems provide sustained release of proteins, thereby extending the efficacy of such therapies. Here, we investigated the biocompatibility of a novel coacervate delivery system based on poly(ethylene argininylaspartate diglyceride) (PEAD) and heparin in the damaged spinal cord. We found that the presence of the [PEAD:heparin] coacervate did not affect the macrophage response, glial scarring, or nervous tissue loss, which are hallmarks of spinal cord injury. Moreover, the density of axons, including serotonergic axons, at the injury site and the recovery of motor and sensorimotor function were comparable in rats with and without the coacervate. These results revealed the biocompatibility of our delivery system and supported its potential to deliver therapeutic proteins to the injured nervous system.

Cell therapy for nervous tissue repair is limited by low transplant survival. We investigated the effects of a polyurethane-based reverse thermal gel, poly(ethylene glycol)–poly(serinol hexamethylene urethane) (ESHU) on bone marrow stromal cell (BMSC) transplant survival and repair using a rat model of spinal cord contusion. Transplantation of BMSCs in ESHU at three days post-contusion resulted in a 3.5-fold increase in BMSC survival at one week post-injury and a 66% increase in spared nervous tissue volume at four weeks post-injury. These improvements were accompanied by enhanced hindlimb motor and sensorimotor recovery. In vitro, we found that ESHU protected BMSCs from hydrogen peroxide-mediated death, resulting in a four-fold increase in BMSC survival with two-fold fewer BMSCs expressing the apoptosis marker, caspase 3 and the DNA oxidation marker, 8-oxo-deoxyguanosine. We argue that ESHU protected BMSCs transplanted is a spinal cord contusion from death thereby augmenting their effects on neuroprotection leading to improved behavioral restoration. The data show that the repair effects of intraneural BMSC transplants depend on the degree of their survival and may have a widespread impact on cell-based regenerative medicine.

PURPOSE We assessed the in vivo release profile of bevacizumab from and biocompatibility of poly(ethylene glycol)-poly-(serinol hexamethylene urethane), or ESHU, a thermoresponsive hydrogel administered intravitreally for drug delivery. METHODS The technical feasibility of injection was assessed quantitatively via mechanical testing. For in vivo studies, New Zealand White rabbit eyes were injected intravitreally with 0.05 mL of either: ESHU dissolved in 25 mg/mL bevacizumab, ESHU dissolved in PBS, or 25 mg/mL bevacizumab. Clinical examination included IOP measurements and examination with indirect ophthalmoscopy for signs of inflammation. Additionally, eyes were examined histologically following euthanasia. To quantify bevacizumab release, aqueous humor samples were obtained via anterior chamber paracentesis and ELISA was used to determine the concentration of drug weekly. In vitro cytotoxicity testing also was performed using bovine corneal endothelial cells. RESULTS The ESHU was injected easily through a 31-gauge needle, was well tolerated in vivo, and caused minimal cell death in vitro when compared to other common materials, such as silicone oil. The long-term presence of the gel did not affect IOP, and there was no evidence of inflammation histologically or through indirect observation. The ESHU sustained the release of bevacizumab for over 9 weeks and maintained a drug concentration that averaged 4.7 times higher than eyes receiving bolus bevacizumab injections. CONCLUSIONS To our knowledge, this is the first report demonstrating sustained bevacizumab release in vivo from an intravitreally injected hydrogel formulation, suggesting that this delivery system may be a promising candidate for ocular drug delivery.

After traumatic spinal cord injury (SCI), there is an opportunity for preserving function by attenuating secondary cell loss. Astrocytes play crucial roles in the adult CNS and are responsible for the vast majority of glutamate buffering, potentially preventing excitotoxic loss of neurons and oligodendrocytes. We examined spatial and temporal changes in gene expression of the major astrocyte glutamate transporter GLT1 following moderate thoracic contusion SCI using transgenic BAC-GLT1-eGFP promoter reporter mice. In dorsal column white matter, total intensity of GLT1-eGFP expression per region was significantly reduced following SCI at both lesion epicenter and at rostral and caudal areas where no tissue loss occurred. This regional decrease in GLT1 expression was due to significant loss of GLT1-eGFP(+) cells, partially accounted for by apoptosis of eGFP(+) /GFAP(+) astrocytes in both white and gray matter. There were also decreased numbers of GLT1-eGFP-expressing cells in multiple gray matter regions following injury; nevertheless, there was sustained or even increased regional GLT1-eGFP expression in gray matter as a result of up-regulation in astrocytes that continued to express GLT1-eGFP. Although there were increased numbers of GFAP(+) cells both at the lesion site and in surrounding intact spinal cord following SCI, the majority of proliferating Ki67(+) /GFAP(+) astrocytes did not express GLT1-eGFP. These findings demonstrate that spatial and temporal alterations in GLT1 expression observed after SCI result from both astrocyte death and gene expression changes in surviving astrocytes. Results also suggest that following SCI a significant portion of astrocytes lacks GLT1 expression, possibly compromising the important role of astrocytes in glutamate homeostasis.

Cellular abnormalities in amyotrophic lateral sclerosis (ALS) are not limited to motor neurons. Astrocyte dysfunction also occurs in human ALS and transgenic rodents expressing mutant human SOD1 protein (SOD1(G93A)). Here we investigated focal enrichment of normal astrocytes using transplantation of lineage-restricted astrocyte precursors, called glial-restricted precursors (GRPs). We transplanted GRPs around cervical spinal cord respiratory motor neuron pools, the principal cells whose dysfunction precipitates death in ALS. GRPs survived in diseased tissue, differentiated efficiently into astrocytes and reduced microgliosis in the cervical spinal cords of SOD1(G93A) rats. GRPs also extended survival and disease duration, attenuated motor neuron loss and slowed declines in forelimb motor and respiratory physiological functions. Neuroprotection was mediated in part by the primary astrocyte glutamate transporter GLT1. These findings indicate the feasibility and efficacy of transplantation-based astrocyte replacement and show that targeted multisegmental cell delivery to the cervical spinal cord is a promising therapeutic strategy for slowing focal motor neuron loss associated with ALS.

Current clinical therapies have limited capacity to restore function in damaged nerves. Regeneration of axons in the injured central nervous system is inhibited by the presence of both physical and mechanical barriers. As such, neural tissue engineering strategies have received considerable attention in recent years. Materials-based approaches offer a way to blend synthetic and biological components in order to generate environments conducive to neural regeneration. While biomaterials themselves can facilitate regeneration simply by providing a substrate for cell growth, incorporation of “bioactive cues”—or molecules which interact with cells to elicit a specific response—has become increasingly necessary in order to promote nerve regeneration. This review attempts to outline the various biomaterials that are promising for central nerve applications, and discusses the array of bioactive cues that are valuable in promoting nerve repair.