Use of Bromodeoxyuridine Immunohistochemistry for the Study of Cytogenesis and Neurochemical and Cellular Differentiation in the Marsupial Brain and Retina

Determining the birthdate of neurons and glia in relation lO the time period of neurochemical 1.nd cellular differentiation are necessary for understanding early developmental events in the central nerv.\u3eus system (CNS). Bromodeoxyuridine (BrdU). a thymidine analog has recently become an alternative to the tritiated thymidine autoradiography for the study of cytogenesis in the CNS. Our laboratory utilizes the Brazilian opossum, Monodelphis domestica to study the ontogeny of neuropeptide systems and the development of the visual system in the mammalian brain. Opossum pups are born in an extremely immature state before neurogenesis and morphogenesis are completed and provide an excellent experimental model for developmental studies. To this end, we have developed single, double. and triple label BrctU immunohistochemical procedures for simultaneous study of cytogenesis and cellular differentiation in the opossum brain and retina. These methods are simple, rapid, and very effective and could be widely applied for understanding developmental events in the CNS

Bone marrow-derived mesenchymal stem cells (MSCs) stimulate neurite outgrowth from differentiating adult hippocampal progenitor cells

Background: Bone marrow-derived mesenchymal stem cells (MSCs) have emerged as beneficial cellular vehicles for nervous system rescue and repair. A better understanding how MSCs are involved in mediating neural repair will facilitate development of novel therapeutic strategies. Methods: In the present study bone marrow-derived MSCs were isolated and characterized from Brown Norway rats. The capacity of the MSCs to influence the differentiation of adult hippocampal progenitor cells (AHPCs) was investigated using contact and non-contact co-culture configurations. Results: These MSCs showed a stable and consistent growth rate, retained short population doubling time (PDT) and showed high capacity of cell proliferation. Co-culturing of AHPCs with MSCs did not appear to significantly affect the proliferation of the AHPCs or impact the proportion of neuronal or glial differentiation of the AHPCs. However, both contact co-culture (CCC) and non-contact co-culture (NCCC) significantly promoted neurite outgrowth from neuronal AHPCs. Conclusions: The ability of MSCs to promote the morphological differentiation of AHPCs may serve as an added benefit when developing cell-based strategies for nervous system rescue and repair

Recovery of canine retina and optic nerve function after acute elevation of intraocular pressure: implications for canine glaucoma treatment

Purpose  To characterize the timing and extent of functional recovery in healthy canine eyes exposed to acute elevation of intraocular pressure (IOP). Methods  Acute elevation of IOP was induced in 14 healthy Beagles by elevating IOP above the levels of systolic blood pressure for 60 min (average elevation was between 100 and 160 mmHg). Menace, dazzle and pupillary light reflexes (PLR) were tested at 1, 7, 14 and 28 days post elevation. Optical coherence tomography was used to evaluate retinal thickness preoperatively and at 15 and 30 days post elevation. Results  One day post elevation all animals were blind in the operated eye (no positive menace), 5/14 had positive PLR and 10/14 had positive dazzle response. Seven days post elevation 4/14 animals had positive menace response and all animals (14/14) had positive dazzle and PLR responses. Fourteen and 28 days post elevation all animals had positive menace, PLR and dazzle responses. Optical coherence tomography analysis revealed significant thinning of the inferior retina (pre elevation: 156.3 ± 4.8 µm; 15 days post elevation: 125 ± 10.4 µm; 30 days post elevation 123 ± 11.9 µm; P \u3c 0.01, anova). The superior retina, however, did not show any detectable decrease in thickness compared to control eyes (pre elevation: 193.8 ± 2.6 µm; 15 days post elevation: 176.9 ± 8.5 µm; 30 days post elevation 176.9 ± 7 µm; P = 0.057, anova). Conclusions  Detailed functional and morphologic analysis revealed precise information about retinal damage after acute elevation of IOP. Canine retina has the capacity to recover at least some visual function even at 14 days after acute elevation of the IOP. More aggressive medical and surgical treatment of canine glaucomatous patients may be indicated despite complete loss of visual function, PLR and dazzle responses in early days after development of an acute glaucomatous attack

Functional Characterization of Retina and Optic Nerve after Acute Ocular Ischemia in Rats

purpose. To functionally characterize the status of the rat retina and optic nerve after acute elevation of intraocular pressure (IOP) and to determine the dynamics of the pathologic changes in the ischemic retina and optic nerve. methods. Retinal ischemia was induced in rats by acutely increasing the IOP (110 mm Hg/60 minutes). Direct and indirect pupil light reflexes (PLRs) were recorded from the noninjured eye, and electroretinograms (flash and flicker ERG) were recorded from the injured and control eyes before and after surgery. Amplitudes and latencies were calculated for each recording session. results. Preoperative PLRratios (indirect/direct PLR) were 76.7 ± 2.6 (mean ± SEM). Twenty-four hours after surgery the PLRratio was 15.2 ± 12.8, 10 days after surgery, 11.6 ± 9.8; 20 days after surgery, 26.5 ± 8.0; and 28 days after surgery, 33.27 ± 9.3. However, at day 35, the PLR had significantly recovered (41.1 ± 7.3) when compared with the 24-hour postoperative ratios (P \u3c 0.01, repeated-measures ANOVA). Forty-two days after surgery, the PLRratio started to decrease once again in the injured eyes (28.7 ± 5.9). Electroretinographic amplitudes (full-field flash ERG) followed a similar pattern. Cone responses (flicker ERG) were measured 42 days after surgery and revealed defects in injured eyes (control eyes: 46.6 ± 2.9 μV, injured eyes: 3.4 ± 1.7 μV). Histologic analysis revealed ischemic damage to all retinal layers, with the primary defects localized to the central retina. conclusions. Acute ocular ischemia causes a significant decrease in retinal function, as measured by PLR and ERG, although over time the rat retina and optic nerve show partial regain of function

Experimental study using multiple strains of prion disease in cattle reveals an inverse relationship between incubation time and misfolded prion accumulation, neuroinflammation and autophagy

Proteinopathies result from aberrant folding and accumulation of specific proteins. Currently, there is a lack of knowledge about the factors that influence disease progression making this a key challenge for the development of therapies for proteinopathies. Due to the similarities between transmissible spongiform encephalopathies (TSEs) and other protein misfolding diseases, TSEs can be used to understand other proteinopathies. Bovine spongiform encephalopathy (BSE) is a TSE that occurs in cattle and can be subdivided into three strains: classical BSE, and atypical BSEs (H-type and L-type) that have shorter incubation periods. The NLRP3 inflammasome is a critical component of the innate immune system that leads to release of IL-1β (Interlukin-1β). Macroautophagy is an intracellular mechanism that plays an essential role in protein clearance. In this study, we use the retina as a model to investigate the relationship between disease incubation period, prion protein (PrPSc) accumulation, neuroinflammation, and changes in macroautophagy. We demonstrate that atypical BSEs present with increased PrPSc accumulation and neuroinflammation, and decreased autophagy. Our work suggests a relationship between disease time course, neuroinflammation, and the autophagic stress response. This work may help identify novel therapeutic biomarkers that can delay or prevent the progression of proteinopathies

Multipotent adult hippocampal progenitor cells maintained as neurospheres favor differentiation toward glial lineages

Adult hippocampal progenitor cells (AHPCs) are generally maintained as a dispersed monolayer population of multipotent neural progenitors. To better understand cell-cell interactions among neural progenitors and their influences on cellular characteristics, we generated free-floating cellular aggregates, or neurospheres, from the adherent monolayer population of AHPCs. Results from in vitro analyses demonstrated that both populations of AHPCs were highly proliferative under maintenance conditions, but AHPCs formed in neurospheres favored differentiation along a glial lineage and displayed greater migrational activity, than the traditionally cultured AHPCs. To study the plasticity of AHPCs from both populations in vivo, we transplanted GFP-expressing AHPCs via intraocular injection into the developing rat eyes. Both AHPC populations were capable of surviving and integrating into the developing host central nervous system, but considerably more GFP-positive cells were observed in the retinas transplanted with neurosphere AHPCs, compared to adherent AHPCs. These results suggest that the culture configuration during maintenance for neural progenitor cells (NPCs) influences cell fate and motility in vitro as well as in vivo. Our findings have implication for understanding different cellular characteristics of NPCs according to distinct intercellular architectures and for developing cell-based therapeutic strategies using lineage-committed NPCs

Lasting Retinal Injury in a Mouse Model of Blast-Induced Trauma

Traumatic brain injury due to blast exposure is currently the most prevalent of war injuries. Although secondary ocular blast injuries due to flying debris are more common, primary ocular blast exposure resulting from blast wave pressure has been reported among survivors of explosions, but with limited understanding of the resulting retinal pathologies. Using a compressed air-driven shock tube system, adult male and female C57BL/6 mice were exposed to blast wave pressure of 300 kPa (43.5 psi) per day for 3 successive days, and euthanized 30 days after injury. We assessed retinal tissues using immunofluorescence for glial fibrillary acidic protein, microglia-specific proteins Iba1 and CD68, and phosphorylated tau (AT-270 pThr181 and AT-180 pThr231). Primary blast wave pressure resulted in activation of Müller glia, loss of photoreceptor cells, and an increase in phosphorylated tau in retinal neurons and glia. We found that 300-kPa blasts yielded no detectable cognitive or motor deficits, and no neurochemical or biochemical evidence of injury in the striatum or prefrontal cortex, respectively. These changes were detected 30 days after blast exposure, suggesting the possibility of long-lasting retinal injury and neuronal inflammation after primary blast exposure

High Throughput Characterization of Adult Stem Cells Engineered for Delivery of Therapeutic Factors for Neuroprotective Strategies

Mesenchymal stem cells (MSCs) derived from bone marrow are a powerful cellular resource and have been used in numerous studies as potential candidates to develop strategies for treating a variety of diseases. The purpose of this study was to develop and characterize MSCs as cellular vehicles engineered for delivery of therapeutic factors as part of a neuroprotective strategy for rescuing the damaged or diseased nervous system. In this study we used mouse MSCs that were genetically modified using lentiviral vectors, which encoded brain-derived neurotrophic factor (BDNF) or glial cell-derived neurotrophic factor (GDNF), together with green fluorescent protein (GFP). Before proceeding with in vivo transplant studies it was important to characterize the engineered cells to determine whether or not the genetic modification altered aspects of normal cell behavior. Different culture substrates were examined for their ability to support cell adhesion, proliferation, survival, and cell migration of the four subpopulations of engineered MSCs. High content screening (HCS) was conducted and image analysis performed. Substrates examined included: poly-L-lysine, fibronectin, collagen type I, laminin, entactin-collagen IV-laminin (ECL). Ki67 immunolabeling was used to investigate cell proliferation and Propidium Iodide staining was used to investigate cell viability. Time-lapse imaging was conducted using a transmitted light/environmental chamber system on the high content screening system. Our results demonstrated that the different subpopulations of the genetically modified MSCs displayed similar behaviors that were in general comparable to that of the original, non-modified MSCs. The influence of different culture substrates on cell growth and cell migration was not dramatically different between groups comparing the different MSC subtypes, as well as culture substrates. This study provides an experimental strategy to rapidly characterize engineered stem cells and their behaviors before their application in longterm in vivo transplant studies for nervous system rescue and repair

Evaluation of retinal status using chromatic pupil light reflex activity in healthy and diseased canine eyes

purpose. To differentiate rod-cone–mediated pupil light reflexes (PLRs) from intrinsic melanopsin-mediated pupil light reflexes by comparing pupil responses with red and blue light stimuli of differing intensities in normal dog eyes and in those with sudden acquired retinal degeneration syndrome (SARDS) exhibiting a nonrecordable electroretinogram. methods. The PLR was evaluated in 14 healthy dogs using a computerized pupillometry system and in five dogs with SARDS. Contraction amplitude, velocity, and implicit time of the PLR were studied as a function of peak wavelength (480 nm vs. 630 nm) and light intensity (−0.29 to 5.3 log units) to determine characteristics of the rod-cone versus predominantly melanopsin-mediated PLR activity. results. The PLR in healthy, mildly sedated dogs could be elicited at low light intensities (−0.29 log units; 0.51 cd/m2). Canine SARDS patients displayed a complete absence of vision, electroretinographic amplitude, and PLR at low light intensity. However, in SARDS dogs, a pupil light reflex could be elicited with wavelengths corresponding to the melanopsin spectral sensitivity (blue light − peak at 480 nm) and at relatively high intensity (4.3 log units or higher), whereas red light (630 nm peak wavelength) was ineffective in eliciting any detectable PLR response even at light intensities of 6 log units (1,000,000 cd/m2). conclusions. The PLR in healthy canine eyes can be elicited at very low light intensities using red and blue wavelengths of light, but in dogs with blindness caused by SARDS, the pupil reacts only to high-intensity blue wavelength light, implying loss of the rod-cone–mediated PLR and most likely the presence of intrinsic, melanopsin-mediated, retinal ganglion cell–mediated PLR.This article is from Investigative Ophthalmology & Visual Science 48 (2007): 5178, doi: 10.1167/iovs.07-0249.</p