Biomechanical investigation of a customized interspinous spacer system in the treatment of degenerative disc diseases: A finite element analysis.

BACKGROUND: A novel interspinous fixation system based on anatomical parameters and incorporating transfacetopedicular screws, was developed to treat degenerative disc diseases. The biomechanical characteristics of the novel system were evaluated using finite element analysis in comparison to other classical interspinous spacers. METHODS: The L1-S1 lumbar spine finite element models were surgically implanted with the novel system, Coflex and DIAM devices at the L4/L5 segment to assess the range of motion, the pression distribution of intervertebral disc, the peak stresses on the spinous process and implant during various motions. FINDINGS: Range of motions of the L4/L5 surgical segment were reduced by 29.13%, 61.27%, 77.35%, 33.33%, and the peak stresses of intervertebral disc were decreased by 36.82%, 67.31%, 73.00%, 69.57% for the novel system in flexion, extension, lateral bending, and axial rotation when compared with the Coflex, and they were declined by 34.53%, 57.86%, 75.81%, 25.21%; 36.22%, 67.31%, 75.01%, 71.40% compared with DIAM. The maximum stresses of the spinous process were 29.93 MPa, 24.66 MPa, 14.45 MPa, 24.37 MPa in the novel system, and those of Coflex and DIAM were 165.3 MPa, 109 MPa, 84.79 MPa, 47.66 MPa and 52.59 MPa, 48.78 MPa, 50.27 MPa, 44.16 MPa during the same condition. INTERPRETATION: Compared to other interspinous spacer devices, the novel interspinous fixation system demonstrated excellent stability, effectively distributing load on the intervertebral disc, and reducing the risk of spinous process fractures. The personalized design of the novel interspinous fixation system could be a viable option for treating degenerative disc diseases.

Biomechanical effects of osteoporosis severity on the occurrence of proximal junctional kyphosis following long-segment posterior thoracolumbar fusion.

BACKGROUND: Proximal junctional kyphosis is a common long-term complication in adult spinal deformity surgery that involves long-segment posterior spinal fusion. However, the underlying biomechanical mechanisms of the impact of osteoporosis on proximal junctional kyphosis remain unclear. The present study was to evaluate adjacent segment degeneration and spine mechanical instability in osteoporotic patients who underwent long-segment posterior thoracolumbar fusion. METHODS: Finite element models of the thoracolumbar spine T1-L5 with posterior long-segment T8-L5 fusion under different degrees of osteoporosis were constructed to analyze intervertebral disc stress characterization, vertebrae mechanical transfer, and pedicle screw system loads during various motions. FINDINGS: Compared with normal bone mass, the maximum von Mises stresses of T7 and T8 were increased by 20.32%, 22.38%, 44.69%, 4.49% and 29.48%, 17.84%, 40.95%, 3.20% during flexion, extension, lateral bending, and axial rotation in the mild osteoporosis model, and by 21.21%, 18.32%, 88.28%, 2.94% and 37.76%, 15.09%, 61.47%, -0.04% in severe osteoporosis model. The peak stresses among T6/T7, T7/T8, and T8/T9 discs were 14.77 MPa, 11.55 MPa, and 2.39 MPa under lateral bending conditions for the severe osteoporosis model, respectively. As the severity of osteoporosis increased, stress levels on SCR8 and SCR9 intensified during various movements. INTERPRETATION: Osteoporosis had an adverse effect on proximal junctional kyphosis. The stress levels in cortical bone, intervertebral discs and screws were increased with bone mass loss, which can easily lead to intervertebral disc degeneration, bone destruction as well as screw pullout. These factors have significantly affected or accelerated the occurrence of proximal junctional kyphosis.

Ex vivo MRI facilitates localization of cerebral microbleeds of different ages during neuropathology assessment.

Cerebral microbleeds (CMBs) identified by in vivo magnetic resonance imaging (MRI) of brains of older persons may have clinical relevance due to their association with cognitive impairment and other adverse neurologic outcomes, but are often not detected in routine neuropathology evaluations. In this study, the utility of ex vivo MRI in the neuropathological identification, localization, and frequency of CMBs was investigated. The study included 3 community dwelling elders with Alzheimer's dementia, and mild to severe small vessel disease (SVD). Ex vivo MRI was performed on the fixed hemisphere to identify CMBs, blinded to the neuropathology diagnoses. The hemibrains were then sliced at 1 cm intervals and 2, 1 or 0 microhemorrhages (MH) were detected on the cut surfaces of brain slabs using the routine neuropathology protocol. Ex vivo imaging detected 15, 14 and 9 possible CMBs in cases 1, 2 and 3, respectively. To obtain histological confirmation of the CMBs detected by ex vivo MRI, the 1 cm brain slabs were dissected further and MHs or areas corresponding to the CMBs detected by ex vivo MRI were blocked and serially sectioned at 6 µm intervals. Macroscopic examination followed by microscopy post ex vivo MRI resulted in detection of 35 MHs and therefore, about 12 times as many MHs were detected compared to routine neuropathology assessment without ex vivo MRI. While microscopy identified previously unrecognized chronic MHs, it also showed that MHs were acute or subacute and therefore may represent perimortem events. Ex vivo MRI detected CMBs not otherwise identified on routine neuropathological examination of brains of older persons and histologic evaluation of the CMBs is necessary to determine the age and clinical relevance of each hemorrhage.

TDP-43 pathology and memory impairment in elders without pathologic diagnoses of AD or FTLD.

OBJECTIVE: To investigate the association of TAR DNA-binding protein 43 (TDP-43) pathology with memory, other cognitive domains, and dementia in community-dwelling elders without pathologic diagnoses of Alzheimer disease (AD) or frontotemporal lobar degeneration (FTLD). METHODS: Of 1,058 autopsied participants, 343 (32.4%) did not have pathologic diagnoses of AD or FTLD. Diagnosis of dementia was based on clinical evaluation and cognitive performance tests, which were used to create summary measures of global cognition and of 5 cognitive domains. TDP-43 pathology evaluated in 6 brain regions by immunohistochemistry was converted into a summary measure of TDP-43 severity. RESULTS: Of 343 participants, 135 (39.4%) had TDP-43 pathology with a mean TDP-43 severity score of 0.394 (SD 0.490). TDP-43 inclusions were confined to the amygdala (stage 1) in 43.7% of participants, 40% showed additional involvement of the hippocampus or entorhinal cortex (stages 2), while fewer (16.3%) showed additional TDP-43 pathology in the temporal and frontal cortices (stage 3). Severity of TDP-43 pathology was independently related to lower function in global cognition and episodic and semantic memory while increased odds of dementia was only a trend. When participants with hippocampal sclerosis (HS) were excluded from the models, TDP-43 pathology remained associated with lower episodic memory but relationships with global cognition, semantic memory, and dementia were attenuated. CONCLUSIONS: TDP-43 pathology in elders, without pathologic diagnoses of AD or FTLD, is common and independently associated with lower function in episodic memory, while its associations with global cognitive impairment and dementia are difficult to separate from HS.

Biglycan Inhibits Capsaicin-Induced Substance P Release by Cultured Dorsal Root Ganglion Neurons.

OBJECTIVE: The purpose of this study was to examine the inhibitory effects of biglycan on substance P release from cultured sensory neurons in response to capsaicin. STUDY DESIGN: In vitro study of cultured primary sensory neurons from the rabbit dorsal root ganglion (DRG). We interrogated the culture system function with capsaicin. Biglycan is an important structural component of the intervertebral disc that may regulate growth factors and inflammatory mediators. We tested the hypothesis that biglycan inhibits substance P release in response to capsaicin. RESULTS: The DRG cultures were shown to contain both neurons and astrocytes by immunostaining using antibodies recognizing neuron and glial cell markers. Cultured DRG cells respond to capsaicin in a dose- and time-dependent manner (capsaicin dose ranges from 5 to 500 µmol/L; stimulation time ranges from 0 to 60 minutes). The neurons preincubated with biglycan released 27% less substance P compared with neurons without biglycan (n = 4, P = 0.036). CONCLUSION: We have established a DRG cell culture system, which contains both sensory neurons and the supporting astrocytes. Biglycan, an inhibitor of substance P release by DRG cultures, may serve as an ingredient in intradiscal injectables to reduce back pain.

[Finite Element Analysis of Biomechanics of Cervical Spine after Dynamic Cervical Implant Surgery].

This study aims to investigate the range of motion(ROM)and the stress variation in the intervertebral disc and the vertebral body on adjacent segments and the influence of force transmission mode after the dynamic cervical implant(DCI)surgery.Two types of surgery,DCI implantation and interbody fusion were used to establish the finite element model of the cervical C5,6segment degeneration treatment.The ROM and the adjacent discs and vertebral body stresses of two procedures under flexion,extension,lateral bending and axial rotation working conditions were analyzed.The results showed that ROM of the surgical segment in DCI model was well preserved and could restore to the normal ROM distributions(reduction of the amplitude was less than 25%),and the kinetic characteristics of adjacent segments was less affected.In fusion surgery model,however,ROM of the surgical segment was reduced by 86%-91%,while ROM,disc stress and vertebral stress of adjacent segments were increased significantly,and stress of the C5 vertebral body was increased up to 171.21%.Therefore DCI surgery has relatively small influence on cervical ROM and stress.The study provides a theoretical basis for DCI and fusion surgery in clinic.

Allogeneic Articular Chondrocyte Transplantation Downregulates Interleukin 8 Gene Expression in the Degenerating Rabbit Intervertebral Disk In Vivo.

OBJECTIVE: The aim of this study was to investigate whether repopulating the degenerating intervertebral disk (IVD) with articular chondrocytes will decrease inflammation in the degenerating rabbit IVD. DESIGN: This was a biologic study in a rabbit IVD-injury model in vivo. Dual cell tracking methods (infrared dye labeling and adenovirus transduction) were used to demonstrate the viability of allogeneic articular chondrocytes injected into degenerating rabbit IVDs. Interleukin 8 gene expression was determined via real-time polymerase chain reaction. Infiltrating inflammatory cells (macrophages, T cells, or neutrophils) were examined with immunohistochemistry. The IVDs were also examined by routine histology. RESULTS: Articular chondrocytes labeled with infrared dye were detected in the degenerating IVDs at both 2 and 8 wks after injection. At the 2-wk time point, interleukin 8 gene expression was comparable in IVDs injected with chondrocytes and in intact disks as control (P = 0.647), whereas its expression in IVDs injected with saline increased 50-fold (P = 0.028). Transgene expression of red fluorescent protein, ß-galactosidase, and human bone morphogenetic protein 7 diminished at 8 wks after injection. IVDs injected with chondrocytes overexpressing human bone morphogenetic protein 7 did not show lower interleukin 8 gene expression or improved histology. Macrophages were consistently detected by immunohistochemistry in the cartilage formed around the needle insertion sites in both the saline and chondrocyte groups, whereas neither T cells nor neutrophils were detected. CONCLUSIONS: Allogeneic rabbit articular chondrocyte survived in the degenerating rabbit IVDs for at least 8 wks. Cell treatment resulted in reduced IVD inflammation but did not significantly improve IVD structure.

Cytotoxicity of local anesthetics and nonionic contrast agents on bovine intervertebral disc cells cultured in a three-dimensional culture system.

BACKGROUND CONTEXT: Carragee et al. reported an accelerated progression of lumbar intervertebral disc (IVD) degeneration after discography in a human trial. Local anesthetics and contrast agents have exhibited toxicity to cardiac, renal, and neuronal cells. We hypothesize that local anesthetics or contrast agents commonly injected into the disc space during discography may result in cytotoxicity in vitro. In this study, we compared the cytotoxicity of these agents, alone or in combination, using nucleus pulposus (NP) and annulus fibrosus (AF) cells in a three-dimensional (3D) culture system. PURPOSE: The purpose of this study was to examine the effects of local anesthetics and contrast agents on IVD cells to help guide their usage in future clinical practices. STUDY DESIGN: Ours was an in vitro study to assess the cytotoxicity of local anesthetics and contrast agents commonly used in discography, using bovine NP and AF cells cultured in a 3D system. METHODS: Bovine NP and AF cells were isolated and encapsulated in alginate beads and cultured in media completed with serum and ascorbic acid. Beads were transferred to a 24-well plate and treated with local anesthetics, nonionic contrast agents, or with saline as a control for 2, 6, and 16 hours. Three different concentrations of local anesthetics, lidocaine and bupivacaine, were tested: 0.25%, 0.125%, and 0.0625%. Two different dilutions (1:2 or 1:4) of nonionic contras agents, iohexol and iopamidol, were tested. In a parallel study, beads were incubated with a combination of local anesthetics at equipotent concentrations and contrast agents for 6 hours. Cells were then examined with the LIVE/DEAD cell assay. Live cells (fluorescing green) and dead cells (fluorescing red) were visualized using fluorescent microscopy. The percentage of live cells after treatment was determined. RESULTS: More cell death was observed when NP and AF cells were incubated with anesthetics than contrast agents at the concentrations tested. When tested at equipotent concentrations, 0.125% bupivacaine (N=8) resulted in significantly more cell death than 0.5% lidocaine (N=6) in NP cells (p<.05). In these studies, cell death caused by bupivacaine was both dose and time dependent. When tested at the same dilutions, iopamidol diluted 1:2 caused slightly more cell death than iohexol. When incubating the cells with a combination of contrast and anesthetic agent, the cytotoxic effects of the anesthetics and contrast agent were not synergistic. In this culture system, AF cells were more sensitive to some of the agents than NP cells. CONCLUSIONS: Cell death was observed when AF and NP cells were incubated in a dose- and time-dependent manner with local anesthetics and contrast agents commonly used for discography. Relative toxicity of these compounds was noted in the order of bupivacaine, lidocaine, iopamidol, and iohexol. Future studies of the effects of these agents in organ culture or animal models are indicated to predict what happens in vivo.

TDP-43 pathology, cognitive decline, and dementia in old age.

IMPORTANCE: Cognitive decline is a leading cause of disability and death in old age but its neurobiological bases are not well understood. OBJECTIVE: To test the hypothesis that transactive response DNA-binding protein 43 (TDP-43) is related to late-life cognitive decline. DESIGN, SETTING, AND PARTICIPANTS: Longitudinal clinical-pathologic cohort study involving more than 40 Catholic groups across the United States. A total of 130 older Catholic nuns, priests, and monks underwent annual clinical evaluations, including detailed cognitive testing, for a mean of 10.1 years prior to death. On neuropathologic examination, we collected semiquantitative measures of TDP-43 pathology, density of neuronal neurofibrillary tangles, area occupied by amyloid-beta plaques, and the presence of alpha-synuclein Lewy bodies from multiple brain regions. Gross and microscopic cerebral infarcts and hippocampal sclerosis were also identified. MAIN OUTCOMES AND MEASURES: Annual rate of change in a previously established composite measure of global cognition during a mean of 10.1 years of annual observation before death. RESULTS: Transactive response DNA-binding protein 43 pathology, ranging from sparse to severe, was identified in 46% of participants and was associated with amyloid plaques, tangles, and hippocampal sclerosis but not neocortical Lewy bodies or cerebral infarcts. After controlling for amyloid plaques, tangles, and hippocampal sclerosis, TDP-43 pathology was associated with more rapid cognitive decline and accounted for nearly as much of the variability in rates of global cognitive decline as did tangles. Transactive response DNA-binding protein 43 pathology had a distinct cognitive profile that differed from other neuropathologic processes (related to decline in episodic and working memory but not in other cognitive domains), and it was elevated in those who developed dementia but not in those with mild cognitive impairment. CONCLUSION AND RELEVANCE: The results suggest that TDP-43 is an important brain pathology underlying cognitive decline and dementia in old age.

Numerical simulation of the inhibitory effect of angiostatin on metastatic tumor angiogenesis and microenvironment.

The present work formulates and analyzes the inhibitory effect of anti-angiogenic factor angiostatin excreted by the primary tumor on metastatic tumor angiogenesis, blood perfusion, and interstitial fluid flow in the tumor microenvironment by means of a numerical experiment. The simulation results demonstrate that angiostatin has an obvious impact on the morphology, growth rate, and the number of branches of microvascular network inside and outside the metastatic tumor, and angiostatin has the capacity to regulate and inhibit the formation of new blood vessels. Heterogeneous blood perfusion, widespread interstitial hypertension, and low convection within the metastatic tumor have obviously improved under the inhibitory effect of angiostatin, which are consistent with physiological observed facts. The simulation results may provide beneficial information and theoretical models for clinical research of anti-angiogenic therapy strategies.

Long-term gonadal hormone treatment and endogenous neurogenesis in the dentate gyrus of the adult female monkey.

Neurogenesis occurs continually throughout life in all mammals and the extent of neurogenesis is influenced by many factors including gonadal hormones. Most research regarding hormones and neurogenesis has been performed on non-primate species. To determine whether gonadal hormones can modulate endogenous neurogenesis in the dentate gyrus (DG) of the hippocampus in non-human primates, ovariectomized (OVX) female rhesus monkeys received continuous, unopposed beta-estradiol (OVX-E-Con), cyclic unopposed beta-estradiol (OVX-E-Cyc), continuous beta-estradiol+cyclic progesterone (OVX-E-Con+P-Cyc), or control (OVX-Veh) treatments. At week 29, all monkeys received BrdU injections for 4 consecutive days, in addition to the ongoing treatment. Twenty days after the last BrdU injection, all animals were sacrificed for tissue collection. In DG of hippocampus, scattered BrdU-ir cells were observed mainly in the subgranular zone (SGZ) and in the granule cell layer and occasionally these BrdU-ir cells in the SGZ formed clusters containing between 2 and 5 cells. In the granule cell layer and SGZ, virtually none of the BrdU-ir cells were either Dcx, a marker of immature neurons, or GFAP positive. However, an occasional BrdU-ir cell was positive for both neuronal marker NeuN or beta III-tubulin. Unbiased stereological analysis of BrdU-ir cells within the SGZ and the granule cell layer of DG revealed that among the experimental groups, there was no significant difference in number of BrdU-ir cells within the SGZ and the granule cell layer of the DG: OVX-E-Con (1801+/-218.7), OVX-E-Cyc (1783+/-415.6), OVX-E-Con+/-P-Cyc (1721+/-229.6), and OVX-Veh (1263+/-106.3), but a trend towards increased BrdU-ir cells was observed in all the experimental groups.

Doublecortin-expressing cells persist in the associative cerebral cortex and amygdala in aged nonhuman primates.

A novel population of cells that express typical immature neuronal markers including doublecortin (DCX+) has been recently identified throughout the adult cerebral cortex of relatively large mammals (guinea pig, rabbit, cat, monkey and human). These cells are more common in the associative relative to primary cortical areas and appear to develop into interneurons including type II nitrinergic neurons. Here we further describe these cells in the cerebral cortex and amygdala, in comparison with DCX+ cells in the hippocampal dentate gyrus, in three age groups of rhesus monkeys: young adult (12.3 +/- 0.2 years, n = 3), mid-age (21.2 +/- 1.9 years, n = 3) and aged (31.3 +/- 1.8 years, n = 4). DCX+ cells with a heterogeneous morphology persisted in layers II/III primarily over the associative cortex and amygdala in all groups (including in two old animals with cerebral amyloid pathology), showing a parallel decline in cell density with age across regions. In contrast to the cortex and amygdala, DCX+ cells in the subgranular zone diminished in the mid-age and aged groups. DCX+ cortical cells might arrange as long tangential migratory chains in the mid-age and aged animals, with apparently distorted cell clusters seen in the aged group. Cortical DCX+ cells colocalized commonly with polysialylated neural cell adhesion molecule and partially with neuron-specific nuclear protein and gamma-aminobutyric acid, suggesting a potential differentiation of these cells into interneuron phenotype. These data suggest a life-long role for immature interneuron-like cells in the associative cerebral cortex and amygdala in nonhuman primates.

Transgene expression, bioactivity, and safety of CERE-120 (AAV2-neurturin) following delivery to the monkey striatum.

Neurturin (NTN) is a neurotrophic factor for dopaminergic neurons that may be therapeutic for patients with Parkinson's disease (PD). As a crucial component in a series of nonclinical translational studies aimed at testing whether CERE-120 should advance into clinical trials in PD subjects, we characterized the expression, bioactivity and safety of CERE-120, an adeno-associated virus type-2 (AAV2) vector encoding NTN, following delivery to the striatum of nonhuman primates. Monkeys received bilateral injections of CERE-120 across a tenfold range of doses (6 x 10(10) to 6 x 10(11) vector genomes per animal) or formulation buffer (FB) control. We report here, for the first time, a dose-related: increase in NTN protein expression within the striatum and substantia nigra (SN) pars compacta of nonhuman primates; increase in nigrostriatal tyrosine hydroxylase (TH), (the rate-limited enzyme for dopamine); and activation of phosphorylated signal-regulated kinase (a common neurotrophic signaling event). Additionally, extensive toxicology testing revealed no adverse effects of CERE-120 on in-life measures, neurotoxicity (in any site throughout the brain) or systemic pathology (in any organ or tissue) across the tenfold range of doses. Collectively, these data provide substantial novel evidence for the potential utility of CERE-120 as a novel treatment for PD and support ongoing clinical trials testing CERE-120 in PD patients.

Upregulation of dorsal horn microglial cyclooxygenase-1 and neuronal cyclooxygenase-2 after thoracic deep muscle incisions in the rat.

BACKGROUND: Plantar hindpaw incision produces hyperalgesia, transient upregulation of cyclooxygenase-2 (COX-2) and prolonged upregulation of cyclooxygenase-1 (COX-1) in rat lumbar spinal cord. Our hypothesis in this study was that a deep thoracic incision causes COX-1 and COX-2 upregulation in the dorsal horn coincident with pain-related behavior, and that specific cell types contribute to this increase in COX expression. METHODS: A left lateral thoracic skin incision was made in anesthetized rats, and superficial and deep muscles were incised. Postoperative pain-related behavior was quantified by recording exploratory rearing. Four and 24 h postsurgery, COX-1 and COX-2 immunohistochemistry, with co-labeling for cell type, were performed on the spinal cord. RESULTS: Deep thoracic muscle incision produced a 42% decrease in rearing compared to sham skin-incision controls at 4 h postsurgery (P = 0.001). There was an increase in both COX-1 and COX-2 immunoreactivity in the thoracic dorsal horn at 4 h postsurgery on the ipsilateral side of surgery animals compared to the ipsilateral side of control animals, contralateral side of surgery animals or contralateral side of control animals. No surgery-induced differences were seen at the lumbar level. At 24 h postsurgery, there was no longer a decrease in rearing, and no surgery-induced differences in COX-1 or COX-2 were seen at any level. At 4 h postsurgery, 96% of COX-1 immunoreactive cells co-localized with microglia and 98% of COX-2 immunoreactive cells co-localized with neurons. CONCLUSIONS: A unilateral deep thoracic wound produces pain-related behavior and, at the same time, ipsilateral upregulation of microglial COX-1 and neuronal COX-2 in the thoracic dorsal horn.

Sensory neurons and fibers from multiple spinal cord levels innervate the rabbit lumbar disc.

OBJECTIVE: To establish the neurotransmission pathway from the lumbar L5/6 intervertebral disc (IVD) to the spinal cord in the rabbit. DESIGN: Fluorogold particles injected into the posterior portion of the rabbit L5/6 IVD were traced by examining gold-positive neurons and fibers in the dorsal root ganglion (DRG) and spinal cord at various root levels. RESULTS: Fluorogold-labeled neurons were observed bilaterally in primary afferent DRG neurons from the L3 through L5 segments; a small number of gold-labeled neurons were found at the L1 level. Fluorogold-labeled neurons were predominantly present in the ipsilateral DRG (the side of the injection) at the L5 level, but they were more equally distributed (on both sides) at the L4 and L3 levels. In the posterior horn of the spinal cord, Fluorogold particles were found in nerve fibers as rostral as the T12 level. CONCLUSIONS: Our study has shown that Fluorogold particles injected into the rabbit L5/6 IVD are taken up by primary sensory neurons in the DRGs and primary sensory fibers in the posterior horn of the spinal cord at multiple levels. This diffuse innervation pattern of the lumbar disc may help explain why discogenic back pain in humans is often poorly localized.

Survival and early differentiation of human neural stem cells transplanted in a nonhuman primate model of stroke.

OBJECT: Neural cell transplantation has been proposed as a treatment after stroke. The purpose of this study was to establish if human neural stem cells (HNSCs) could survive in the nonhuman primate brain after an ischemic event. METHODS: Three adult cynomolgus monkeys received a unilateral occlusion of the M, segment of the right middle cerebral artery (MCA). One week later each animal received five magnetic resonance (MR) image-guided stereotactic intracerebral injections of HNSC neurospheres labeled with bromodeoxyuridine (BrdU) in the areas surrounding the ischemic lesion as defined in T1- and T2-weighted images. On the day of transplantation and throughout the study the monkeys received oral cyclosporine (10 mg/kg twice a day), and plasma levels were monitored routinely. The animals were killed at 45, 75, or 105 days after transplantation. Magnetic resonance images revealed a cortical and subcortical infarction in the MCA distribution area. Postmortem morphological brain analyses confirmed the distribution of the infarcted area seen in the MR images, with loss of tissue and necrosis in the ischemic region. Cells that were positive for BrdU were present in the three experimental monkeys, mainly along injection tracks. Double-label immunofluorescence for BrdU and betaIII-tubulin (a marker of young neurons) revealed colocalization of few HNSCs, most of which were observed outside the immediate injection site. Colocalization with nestin was also observed, indicating an early neural/glial fate. CONCLUSIONS: In a model of stroke in nonhuman primates, HNSCs can survive up to 105 days when transplanted 1 week after an ischemic event and can partly undergo neuronal differentiation.

Galanin fiber hypertrophy within the cholinergic nucleus basalis during the progression of Alzheimer's disease.

Galanin (GAL)-containing fibers enlarge and hyperinnervate remaining cholinergic basal forebrain (CBF) neurons within the anterior nucleus basalis (NB) in late-stage Alzheimer's disease (AD). Whether GAL hypertrophy occurs in the CBF in the prodromal or early stages of AD remains unknown. The present study used GAL immunohistochemistry and an unbiased semiquantitative scoring method to evaluate GAL innervation in the anterior NB of subjects clinically diagnosed as having no cognitive impairment, mild cognitive impairment or early-stage (mild/moderate) AD. There was no difference in GAL fiber staining within the anterior NB across the three clinical groups examined. Furthermore, GAL fiber innervation was not correlated with the number of NB neurons expressing the nerve growth factor receptors p75(NTR) or TrkA or with cortical choline acetyltransferase activity in the same cases. Single-cell gene expression analysis demonstrated that cholinergic NB neurons express mRNA for the GAL receptors GALR1, GALR2 and GALR3, yet the levels of these mRNAs were unchanged across the three diagnostic groups. These observations indicate that GAL hypertrophy within the anterior NB subfield is a late-stage AD response, which may play a role in regulating the cholinergic tone of remaining basocortical projection neurons.

Nigrostriatal dysfunction in familial Alzheimer's disease-linked APPswe/PS1DeltaE9 transgenic mice.

Alzheimer's disease (AD) is often accompanied by extrapyramidal signs attributed to nigrostriatal dysfunction. The association between amyloid deposition and nigrostriatal degeneration is essentially unknown. We showed previously that the striatum and the substantia nigra of transgenic mice harboring familial AD (FAD)-linked APPswe/PS1DeltaE9 mutants exhibit morphological alterations accompanied by amyloid-beta (Abeta) deposition (Perez et al., 2004). In the present study, we further investigated the interaction between Abeta deposition and dopaminergic nigrostriatal dysfunction, by correlating morphological and biochemical changes in the nigrostriatal pathway with amyloid deposition pathology in the brains of 3- to 17-month-old APPswe/PS1DeltaE9 transgenic mice and age-matched wild-type controls. We show that Abeta deposition is pronounced in the striatum of APPswe/PS1DeltaE9 mice at 6 months of age, and the extent of deposition increases in an age-dependent manner. Tyrosine hydroxylase (TH)-positive dystrophic neurites with rosette or grape-like cluster disposition are observed adjacent to Abeta plaques and display multilaminar, multivesicular, and dense-core bodies as well as mitochondria. In addition, an age-dependent increase of TH protein levels are shown in nigral cells in these mutant mice. Using HPLC analysis, we found a reduction in the dopamine metabolite DOPAC in the striatum of these mice. These findings show a close association between amyloid deposition and nigrostriatal pathology and suggest that altered FAD-linked amyloid metabolism impairs, at least in part, the function of dopaminergic neurons.

RNA amplification of bromodeoxyuridine labeled newborn neurons in the monkey hippocampus.

Neurogenesis has been demonstrated in the adult mammalian hippocampus by the immunohistochemical identification of cells co-labeled with the neuronal marker NeuN and bromodeoxyuridine (BrdU), a marker for DNA synthesis. Whether these newly born neurons exhibit a genetic signature similar to that of existing hippocampal cells remains unknown. Recent advances in single cell RNA amplification techniques provide a unique method for profiling the mRNA complement of cells developed during adult neurogenesis. Standard protocols for identifying BrdU-positive cells requires an acid denaturation step that may preclude the amplification of cellular RNA for expression analysis. We first tested whether the BrdU reaction product was visible in monkey hippocampal tissue following treatment with dilutions of HCl (2-0.2 M) or citric acid (1.0-0.1 M). BrdU-labeled cells were visible only in tissue sections treated with 2 M HCl. RNA amplification was not compromised in cells dual-labeled for BrdU and NeuN using the 2 M HCl acid denaturation step. These cells express mRNAs encoding a wide variety of functional protein subclasses including glutamate receptors. The present study demonstrates for the first time that BrdU immunohistochemisty is compatable with gene array technology in the primate hippocampus to evaluate subclasses of genes in newborn neurons.

Human neural stem cell transplants improve motor function in a rat model of Huntington's disease.

The present study investigated the neuroanatomical and behavioral effects of human stem cell transplants into the striatum of quinolinic acid (QA)-lesioned rats. Twenty-four rats received unilateral QA (200 nM/microl) injections into the striatum. One week later, rats were transplanted with stem cells derived from human fetal cortex (12 weeks postconception) that were either 1) pretreated in culture media with the differentiating cytokine ciliary neurotrophic factor (CNTF; n = 9) or 2) allowed to grow in culture media alone (n=7). Each rat was injected with a total of 200,000 cells. A third group of rats (n=8) was given a sham injection of vehicle. Rats transplanted with human stem cells performed significantly better over the 8 weeks of testing on the cylinder test compared with those treated with vehicle (P < or = 0.001). Stereological striatal volume analyses performed on Nissl-stained sections revealed that rats transplanted with CNTF-treated neurospheres had a 22% greater striatal volume on the lesioned side compared with those receiving transplants of untreated neurospheres (P = 0.0003) and a 26% greater striatal volume compared with rats injected with vehicle (P < or = 0.0001). Numerous human nuclei-positive cells were visualized in the striatum in both transplantation groups. Grafted cells were also observed in the globus pallidus, entopeduncular nucleus, and substantia nigra pars reticulata, areas of the basal ganglia receiving striatal projections. Some of the human nuclei-positive cells coexpressed glial fibrillary acidic protein and NeuN, suggesting that they had differentiated into neurons and astrocytes. Taken together, these data demonstrate that striatal transplants of human fetal stem cells elicit behavioral and anatomical recovery in a rodent model of Huntington's disease.