CNS regeneration after chronic injury using a self-assembled nanomaterial and MEMRI for real-time in vivo monitoring.

To speed up the process of central nervous system (CNS) recovery after injury, the need for real-time measurement of axon regeneration in vivo is essential to assess the extent of injury, as well as the optimal timing and delivery of therapeutics and rehabilitation. It was necessary to develop a chronic animal model with an in vivo measurement technique to provide a real-time monitoring and feedback system. Using the framework of the 4 P's of CNS regeneration (Preserve, Permit, Promote and Plasticity) as a guide, combined with noninvasive manganese-enhanced magnetic resonance imaging (MEMRI), we show a successful chronic injury model to measure CNS regeneration, combined with an in vivo measurement system to provide real-time feedback during every stage of the regeneration process. We also show that a chronic optic tract (OT) lesion is able to heal, and axons are able to regenerate, when treated with a self-assembling nanofiber peptide scaffold (SAPNS). FROM THE CLINICAL EDITOR: The authors of this study demonstrate the development of a chronic injury model to measure CNS regeneration, combined with an in vivo measurement system to provide real-time feedback during every stage of the regeneration process. In addition, they determined that chronic optic tract lesions are able to heal with axonal regeneration when treated with a self-assembling nanofiber peptide scaffold (SAPNS).

Enhanced survival of melanopsin-expressing retinal ganglion cells after injury is associated with the PI3 K/Akt pathway.

In the present study, we studied the factors that contribute to the injury-resistant property of melanopsin-expressing retinal ganglion cells (mRGCs). Since phosphatidylinositol-3 kinase (PI3 K)/Akt signaling pathway is one of the well-known pathways for neuronal cell survival, we investigated the survival of mRGCs by applying the PI3 K/Akt specific inhibitors after injury. Two injury models, unilateral optic nerve transection and ocular hypertension, were adopted using Sprague-Dawley rats. Inhibitors of PI3 K/Akt were injected intravitreally following injuries to inhibit the PI3 K/Akt signaling pathway. Retinas were dissected after designated survival time, immunohistochemistry was carried out to visualize the mRGCs using melanopsin antibody and the number of mRGCs was counted. Co-expression of melanopsin and phospho-Akt (pAkt) was also examined. Compared to the survival of non-melanopsin-expressing RGCs, mRGCs showed a marked resistance to injury and co-expressed pAkt. Application of PI3 K/Akt inhibitors decreased the survival of mRGCs after injury. Our previous study has shown that mRGC are less susceptible to injury following the induction of ocular hypertension. In this study, we report that mRGCs were injury-resistant to a more severe type of injury, the optic nerve transection. More importantly, the PI3 K/Akt pathway was found to play a role in maintaining the survival of mRGCs after injury.

Melanopsin-expressing retinal ganglion cells are more injury-resistant in a chronic ocular hypertension model.

PURPOSE: To investigate the survival of melanopsin-expressing retinal ganglion cells (mRGCs) after the induction of chronic ocular hypertension. METHODS: Intraocular pressure (IOP) was elevated in adult Sprague-Dawley rats using an argon laser to photocoagulate the episcleral and limbal veins. IOP was measured with a calibrated tonometer and monitored for a period. Seven days before the animals were killed, a piece of sterile foam soaked with gold fluorescent dye was placed onto the superior colliculus (SC) to label the SC-projecting retinal ganglion cells (scRGCs) retrogradely. mRGCs were visualized by free floating immunohistochemistry on whole-mounted retinas. The number of surviving scRGCs and mRGCs were counted on flatmounted retinas. The branching pattern of dendrites and soma size of mRGCs were examined. RESULTS: An approximately 1.7-fold increase of IOP and a significant loss of scRGCs were found in experimental eyes after laser photocoagulation. However, no significant cell loss or morphologic changes on mRGCs and their dendrites after the induction of chronic ocular hypertension are noticed over a 12-week period. CONCLUSIONS: Although the degeneration of retinal ganglion cells (RGCs) is a major concern in glaucomatous damage, the findings show that mRGCs are less susceptible to death after the induction of chronic ocular hypertension. This result indicates that mRGCs carry some unique properties that are different from those of other subpopulations of RGCs. The immunohistochemistry approach can be used to distinguish easily these mRGCs from other subtypes. This method provides a useful tool to investigate their injury-resistant properties that are informative for the development of effective neuroprotective treatment for glaucoma.

Expression of nicotinamide adenine dinucleotide phosphate-diaphorase in the retina of postnatal golden hamsters deprived of light stimulation.

Nicotinamide Adenine Dinucleotide Phosphate-Diaphorase (NADPH-d) expressing neurons in the retina of golden hamsters have been identified to be a subset of amacrine cells that provide a major source of Nitric Oxide (NO) in retina. This subset of amacrine cells in mouse retina was recently proved to contain the circadian clock gene Per1 (D.Q. Zhang, T. Zhou, G.X. Ruan, D.G. McMahon, Circadian rhythm of Period 1 clock gene expression in NOS amacrine cells of the mouse retina, Brain Res., 1050 (2005) 101-109). However, it remains unknown whether these clock-related NADPH-d amacrine cells can be regulated by light stimulation and thus synchronized to ambient day/night cycle. A previous study has reported that NADPH-d expressing amacrine cells in postnatal hamsters exhibited a surge after eye-opening (D. Tay, Y.C. Diao, Y.M. Xiao, K.F. So, Postnatal development of nicotinamide adenine dinucleotide phosphate-diaphorase-positive neurons in the retina of the golden hamster, J. Comp. Neurol., 446 (2002) 342-348) suggesting a possible effect of light on the NADPH-d amacrine cells. In order to further reveal the relationship between NADPH-d amacrine cells and light stimulation, the present study focuses on the changes of the expression of NADPH-d in the retina of postnatal hamsters reared in completely deprived light conditions. Prior to eye opening, P12 hamster pups were subjected to either bilateral eyelid suturing or dark rearing. On P28 a subgroup of light deprived hamsters was returned to lighting conditions and the expression of NADPH-d activities in the retina was assessed. In hamsters reared in the 12:12 light-dark cycle, the number of NADPH-d amacrine cells in the ganglion cell layer (GCL) increased right after eye-opening and reached the adult level gradually. However, hamsters subjected to both bilateral eyelid suturing and dark rearing, the number of NADPH-d amacrine cells in GCL was maintained at a low level but increased again upon returning to the 12:12 light-dark condition. In contrast, the number of NADPH-d expressing amacrine cells in the inner nuclear layer (INL) remained low and unaltered regardless of the lighting environment. This study demonstrates that there are two subpopulations of NADPH-d expressing amacrine cells with respect to different locations in the retina of hamsters. Different from those in INL, the NADPH-d amacrine cells in GCL of postnatal hamsters are dependent on the lighting environment implicating that these clock-related amacrine cells and the production of NO might be under a modulation of light stimulation.

Light delays synaptic deafferentation and potentiates the survival of axotomized retinal ganglion cells.

Knowledge of the cellular mechanism underlying the therapeutic effect of stimulation and the optimal doses of such stimulation to maximize neuronal recovery is essential to guide clinical practice in neural rehabilitation. Using hamsters, we transected the optic nerve to demonstrate how light stimulation affects neuronal recovery. The c-fos protein was used as a neuronal connectivity marker. Here we show that: (a) in addition to cell death, a population of cells undergoes synaptic deafferentation and (b) light stimulation delays cell death and deafferentation. Among the three rearing conditions studied (6:18LD, 12:12LD, and 18:6LD), the 12:12LD condition appears to be the one achieving the optimal therapeutic effect. This study provides a solid base in the understanding of the neuroanatomical changes after traumatic brain injury and the need to establish an optimal level and timing for the environmental stimulation.

Nano hemostat solution: immediate hemostasis at the nanoscale.

Hemostasis is a major problem in surgical procedures and after major trauma. There are few effective methods to stop bleeding without causing secondary damage. We used a self-assembling peptide that establishes a nanofiber barrier to achieve complete hemostasis immediately when applied directly to a wound in the brain, spinal cord, femoral artery, liver, or skin of mammals. This novel therapy stops bleeding without the use of pressure, cauterization, vasoconstriction, coagulation, or cross-linked adhesives. The self-assembling solution is nontoxic and nonimmunogenic, and the breakdown products are amino acids, which are tissue building blocks that can be used to repair the site of injury. Here we report the first use of nanotechnology to achieve complete hemostasis in less than 15 seconds, which could fundamentally change how much blood is needed during surgery of the future.

The spider effect: morphological and orienting classification of microglia in response to stimuli in vivo.

The different morphological stages of microglial activation have not yet been described in detail. We transected the olfactory bulb of rats and examined the activation of the microglial system histologically. Six stages of bidirectional microglial activation (A) and deactivation (R) were observed: from stage 1A to 6A, the cell body size increased, the cell process number decreased, and the cell processes retracted and thickened, orienting toward the direction of the injury site; until stage 6A, when all processes disappeared. In contrast, in deactivation stages 6R to 1R, the microglia returned to the original site exhibiting a stepwise retransformation to the original morphology. Thin highly branched processes re-formed in stage 1R, similar to those in stage 1A. This reverse transformation mirrored the forward transformation except in stages 6R to 1R: cells showed multiple nuclei which were slowly absorbed. Our findings support a morphologically defined stepwise activation and deactivation of microglia cells.

Using self-assembled nanomaterials to inhibit the formation of metastatic cancer stem cell colonies in vitro.

The isolation of cells with stem-like properties from prostate tumors suggests the presence of a cancer stem cell (CSC) population, which may account for the initiation, progression, and metastasis as well as drug resistance of the disease. We hypothesized that containing, or at least immobilizing, the CSCs in a nano-self-assembling material might help prevent prostate tumor progression or metastasis. CSCs were plated in three conditions: 1) placed in 1% concentration self-assembled peptide (SAP) preequilibrate with culture medium; 2) placed in 3% concentration SAP preequilibrate with culture medium; and 3) in nonadherent culture. All were grown for 14 days, after which the cells in both 1% and 3% concentrations were washed out of the SAP and grown for an additional 14 days. Here we report that CSCs from prostate cancer cell lines remained quiescent for more than 28 days when embedded in SAP. When the prostate CSCs were embedded in 1% and 3% SAP, most of the CSCs remained single cells 14 days after plating in a nonadherent plate; no prostaspheres could be detected 14 days after plating, suggesting that self-renewal was significantly suppressed. In the controls, prostate CSCs began to divide 1 day after plating in a nonadherent plate and prostaspheres were visible at day 10, indicating the active self-renewal property of the prostate CSCs. Our findings suggest that SAP can completely inhibit a prostate CSC from self-renewal while preserving its viability and CSC property. Therefore, SAP may be an effective nanomaterial for inhibiting cancer progression and metastasis to stop the progression during treatment and removal.

Changes of retinal functions following the induction of ocular hypertension in rats using argon laser photocoagulation.

BACKGROUND: Electroretinography (ERG) provides a longitudinal monitoring of pathological changes in retina. Scotopic threshold response (STR) of ERG was shown to reflect inner retinal activity and is particularly useful in the evaluation of inner retinal changes in ocular hypertension models. Recently, STR was demonstrated to be attenuated after the induction of ocular hypertension using injection of hypertonic saline into episcleral veins which indicates an impairment of retinal function. However, little is known on the changes of retinal function in an ocular hypertension model induced by laser photocoagulation at episcleral veins and limbal veins. METHODS: Ocular hypertension was induced unilaterally using laser photocoagulation at episcleral veins and limbal veins in adult Sprague-Dawley rats. Intraocular pressure was monitored and the number of retinal ganglion cell loss was counted across the 8-week experimental period. The animals were dark adapted overnight and flash ERGs were measured before the laser treatment, 5 and 8 weeks after treatment. The changes of STR, scotopic negative response, a-wave and b-wave were analysed. RESULTS: Approximately 1.6-fold elevation of intraocular pressure was induced in the experimental eyes and 3% retinal ganglion cell loss per week was found. The amplitude of STR was significantly attenuated which indicates an impairment of inner retinal activities. There was also a reduction of scotopic negative response, a-wave and b-wave after the induction of ocular hypertension. CONCLUSION: The laser-induced ocular hypertension model in the present study produced a substantial reduction of retinal functions. Understanding the characteristic of pathological changes is crucial for further study using this model.

Graded ephrin-A2 expression in the developing hamster superior colliculus.

During development, ephrin gradients guide retinal ganglion cell axons to their appropriate topographic locations in the superior colliculus (SC). Expression of ephrin-A2, assessed immunohistochemically in the developing hamster SC, revealed a rostral(low) to caudal (high) gradient that is most prominent at postnatal days P4 and P7 when topography is established. Double-labelling immunohistochemistry for ephrin-A2 and cell specific markers revealed that ephrin-A2 is expressed exclusively by a subset of neurons. The expression pattern has implications for mechanisms underlying establishment of topography during development and following injury.

Nano neuro knitting: peptide nanofiber scaffold for brain repair and axon regeneration with functional return of vision.

Nanotechnology is often associated with materials fabrication, microelectronics, and microfluidics. Until now, the use of nanotechnology and molecular self assembly in biomedicine to repair injured brain structures has not been explored. To achieve axonal regeneration after injury in the CNS, several formidable barriers must be overcome, such as scar tissue formation after tissue injury, gaps in nervous tissue formed during phagocytosis of dying cells after injury, and the failure of many adult neurons to initiate axonal extension. Using the mammalian visual system as a model, we report that a designed self-assembling peptide nanofiber scaffold creates a permissive environment for axons not only to regenerate through the site of an acute injury but also to knit the brain tissue together. In experiments using a severed optic tract in the hamster, we show that regenerated axons reconnect to target tissues with sufficient density to promote functional return of vision, as evidenced by visually elicited orienting behavior. The peptide nanofiber scaffold not only represents a previously undiscovered nanobiomedical technology for tissue repair and restoration but also raises the possibility of effective treatment of CNS and other tissue or organ trauma.

Effects of a therapeutic laser on the ultrastructural morphology of repairing medial collateral ligament in a rat model.

BACKGROUND AND OBJECTIVES: Low energy laser therapy has been shown to enhance mechanical strength of healing medial collateral ligament (MCL) in rats. The present study investigated its effects on the ultrastructural morphology and collagen fibril profile of healing MCL in rats. STUDY DESIGN/MATERIALS AND METHODS: Thirty-two mature male Sprague-Dawley (SD) rats were used. Twenty-four underwent surgical transection to their right MCLs and eight received only skin wound. Immediately after surgery, eight of the MCL transected rats were treated with a single dose of laser therapy at 63.2 J cm(-2), eight were treated with a single dose of laser therapy at 31.6 J cm(-2), the rest had no treatment and served as control. At 3 and 6 weeks after surgery, the MCLs were harvested and examined with electron microscopy for collagen fibril size, distribution, and alignment. RESULTS: Significant differences (P < 0.001) were found in fibril diameters from the same anatomical site and time period among different groups. The mass-averaged diameters of the laser-treated (64.99-186.29 nm) and sham (64.74-204.34 nm) groups were larger than the control group (58.66-85.89 nm). The collagen fibrils occupied 42.55-59.78, 42.63-53.94, and 36.92-71.64% of the total cross-sectional areas in the laser-treated, control and sham groups, respectively. Mode obliquity was 0.53-0.84 among the three groups. CONCLUSIONS: Single application of low energy laser therapy increases the collagen fibril size of healing MCLs in rats.

Therapeutic low energy laser improves the mechanical strength of repairing medial collateral ligament.

BACKGROUND AND OBJECTIVES: Low energy laser therapy has been shown to enhance collagen production but its effect on tissue strength is not well reported. We tested the effects of therapeutic laser on the strength of healing medial collateral ligaments (MCLs) in rats. STUDY DESIGN/MATERIALS AND METHODS: Twenty-four rats received surgical transection to their right MCL and eight received sham operation. After surgery, 16 received a single dose of gallium aluminum arsenide laser to their transected MCL for 7.5 minutes (n = 8) or 15 minutes (n = 8) and eight served as control with placebo laser, while the sham group didn't receive any treatment. The MCLs were biomechanically tested at either 3 or 6 weeks post-operation. RESULTS: The normalized ultimate tensile strength (UTS) and stiffness of laser and sham groups were larger than control (P < 0.001). The UTS of laser and sham groups were comparable. Laser and sham groups had improved in stiffness from 3 to 6 weeks (P < 0.001). CONCLUSIONS: A single dose of low energy laser therapy improves the UTS and stiffness of repairing MCL at 3 and 6 weeks after injury.