Effects of concussion on the blood-brain barrier in humans and rodents.

Traumatic brain injury and the long-term consequences of repeated concussions constitute mounting concerns in the United States, with 5.3 million individuals living with a traumatic brain injury-related disability. Attempts to understand mechanisms and possible therapeutic approaches to alleviate the consequences of repeat mild concussions or traumatic brain injury on cerebral vasculature depend on several aspects of the trauma, including: (1) the physical characteristics of trauma or insult that result in damage; (2) the time "window" after trauma in which neuropathological features develop; (3) methods to detect possible breakdown of the blood-brain barrier; and (4) understanding different consequences of a single concussion as compared with multiple concussions. We review the literature to summarize the current understanding of blood-brain barrier and endothelial cell changes post-neurotrauma in concussions and mild traumatic brain injury. Attention is focused on concussion and traumatic brain injury in humans, with a goal of pointing out the gaps in our knowledge and how studies of rodent model systems of concussion may help in filling these gaps. Specifically, we focus on disruptions that concussion causes to the blood-brain barrier and its multifaceted consequences. Importantly, the magnitude of post-concussion blood-brain barrier dysfunction may influence the time course and extent of neuronal recovery; hence, we include in this review comparisons of more severe traumatic brain injury to concussion where appropriate. Finally, we address the important, and still unresolved, issue of how best to detect possible breakdown in the blood-brain barrier following neurotrauma by exploring intravascular tracer injection in animal models to examine leakage into the brain parenchyma.

Development, differentiation, and vascular components of subcutaneous and intrahepatic Hepa129 tumors in a mouse model of hepatocellular carcinoma.

Tumor models in mice offer opportunities for understanding tumor formation and development of therapeutic treatments for hepatocellular carcinoma. In this study, subcutaneous or intra-hepatic Hepa129 tumors were established in C3H mice. Tumor growth was determined by daily measurements of subcutaneous tumors and post-mortem studies of subcutaneous and intrahepatic tumors. Administration of Edu was used to determine cell generation dates of tumor cells. Immunohistochemistry with antibodies directed at CD31 or CD34, and intravenous injection of labeled tomato lectin revealed tumor vasculature. Tissue sections also were processed for immunohistochemistry using a panel of antibodies to proteoglycans. Comparison of Edu labeled cells with immunoreactivity allowed determination of development and differentiation of tumor cells after cell generation. Subcutaneous and intrahepatic tumors displayed similar growth over 3 weeks. Immunohistochemistry showed strong labeling for glypican-3, 9BA12, and chondroitin sulfate of tumors in both loci, while normal liver was negative. Tumor regions containing Edu labeled cells did not show significant immunohistochemical labeling for the tumor markers until 2-3 days after Edu treatment; overlap of Edu labeled cells and immunohistochemically labeled tumor regions appeared to reach a maximum at 5 days after Edu treatment. Ectopic subcutaneous tumors displayed vascular ingrowth as the tumor cells expressed immunocytochemical markers; subcutaneous tumors displayed significantly more vascular elements than did intrahepatic tumors.

Use of labeled tomato lectin for imaging vasculature structures.

Intravascular injections of fluorescent or biotinylated tomato lectin were tested to study labeling of vascular elements in laboratory mice. Injections of Lycopersicon esculentum agglutinin (tomato lectin) (50-100 µg/100 µl) were made intravascularly, through the tail vein, through a cannula implanted in the jugular vein, or directly into the left ventricle of the heart. Tissues cut for thin 10- to 12-µm cryostat sections, or thick 50- to 100-µm vibratome sections, were examined using fluorescence microscopy. Tissue labeled by biotinylated lectin was examined by bright field microscopy or electron microscopy after tissue processing for biotin. Intravascular injections of tomato lectin led to labeling of vascular structures in a variety of tissues, including brain, kidney, liver, intestine, spleen, skin, skeletal and cardiac muscle, and experimental tumors. Analyses of fluorescence in serum indicated the lectin was cleared from circulating blood within 2 min. Capillary labeling was apparent in tissues collected from animals within 1 min of intravascular injections, remained robust for about 1 h, and then declined markedly until difficult to detect 12 h after injection. Light microscopic images suggest the lectin bound to the endothelial cells that form capillaries and endothelial cells that line some larger vessels. Electron microscopic studies confirmed the labeling of luminal surfaces of endothelial cells. Vascular labeling by tomato lectin is compatible with a variety of other morphological labeling techniques, including histochemistry and immunocytochemistry, and thus appears to be a sensitive and useful method to reveal vascular patterns in relationship to other aspects of parenchymal development, structure, and function.

Neurotrophic factors rescue basal forebrain cholinergic neurons and improve performance on a spatial learning test.

This study investigated whether animals sustaining experimental damage to the basal forebrain cholinergic system would benefit from treatment with exogenous neurotrophic factors. Specifically, we set out to determine whether neurotrophic factors would rescue damaged cholinergic neurons and improve behavioral performance on a spatial learning and memory task. Adult rats received bilateral injections of either saline (controls) or 192 IgG-saporin to damage basal forebrain cholinergic neurons (BFCNs). Two weeks later, animals received implants of an Alzet mini-pump connected to cannulae implanted bilaterally in the lateral ventricles. Animals received infusions of nerve growth factor (NGF), neurotrophin 3 (NT3), a combination of NGF and NT3, or a saline control over a 4-week period. Compared to saline-treated controls, animals sustaining saporin-induced damage to BFCNs took significantly more trials to learn a delayed match to position task and also performed more poorly on subsequent tests, with increasing delays between test runs. In contrast, animals infused with neurotrophins after saporin treatment performed significantly better than animals receiving saline infusions; no differences were detected for performance scores among animals infused with NGF, NT3, or a combination of NGF and NT3. Studies of ChAT immunnocytochemical labeling of BFCNs revealed a reduction in the numbers of ChAT-positive neurons in septum, nucleus of diagonal band, and nucleus basalis in animals treated with saporin followed by saline infusions, whereas animals treated with infusions of NGF, NT3 or a combination of NGF and NT3 showed only modest reductions in ChAT-positive neurons. Together, these data support the notion that administration of neurotrophic factors can rescue basal forebrain cholinergic neurons and improve learning and memory performance in rats.

Characterization of Kupffer cells in livers of developing mice.

BACKGROUND: Kupffer cells are well known macrophages of the liver, however, the developmental characteristics of Kupffer cells in mice are not well understood. To clarify this matter, the characteristics of Kupffer macrophages in normal developing mouse liver were studied using light microscopy and immunocytochemistry. METHODS: Sections of liver tissue from early postnatal mice were prepared using immunocytochemical techniques. The Kupffer cells were identified by their immunoreactivity to the F4/80 antibody, whereas endothelial cells were labelled with the CD-34 antibody. In addition, Kupffer cells and endothelial cells were labelled by systemically injected fluorescently labelled latex microspheres. Tissue slices were examined by fluorescence microscopy. RESULTS: Intravenous or intraperitonal injections of microspheres yielded similar patterns of liver cell labelling. The F4/80 positive Kupffer cells were labelled with both large (0.2 µm) and small (0.02 µm) diameter microspheres, while endothelial cells were labelled only with the smaller diameter microspheres. Microsphere labelling of Kupffer cells appeared stable for at least 6 weeks. Cells immunoreactive for F4/80 were identified as early as postnatal day 0, and these cells also displayed uptake of microspheres. Numbers of F4/80 Kupffer cells, relative to numbers of albumin positive hepatocytes, did not show a significant trend over the first 2 postnatal weeks. CONCLUSIONS: Kupffer cells of the developing mouse liver appear quite similar to those of other mammalian species, confirming that the mouse presents a useful animal model for studies of liver macrophage developmental structure and function.

Binding patterns of peptide-containing liposomes in liver and spleen of developing mice: comparison with heparan sulfate immunoreactivity.

BACKGROUND: Liposomes incorporating peptide from the Plasmodium circumsporozoite protein (CSP) accumulate rapidly and selectively in adult mouse liver. PURPOSE: The development of the liposome-binding pattern in liver and spleen was studied in relationship to the development of extracellular matrix molecules. METHODS: Liposomes were administered to mice intravascularly or applied to the surface of liver and spleen slices in vitro. Slices were analyzed immunocytochemically. RESULTS: Liposomes were found along sinusoidal borders of liver, including the basolateral border of hepatocytes. The pattern was detected in the youngest animals studied (newborn). Intensity of heparan sulfate immunoreactivity increased until adult levels were reached at 20 days. Immunoreactivity for heparan sulfate proteoglycan, but not other proteoglycans, was detected in the youngest animals, and mimicked the pattern of liposome binding. The pattern of liposome binding in the spleen, concentrated in marginal zones, was similar to the pattern of heparan sulfate immunoreactivity, and also similar to the distribution of macrophage immunoreactivity. CONCLUSION: The postnatal development of liposome binding parallels the development of heparan sulfate immunoreactivity, supporting the suggestion that peptide-containing liposomes target liver by binding to heparan sulfate proteoglycans. Specific delivery of liposomes by targeting heparan sulfate proteoglycans is an effective strategy even at early time periods.

Liposomal delivery of doxorubicin to hepatocytes in vivo by targeting heparan sulfate.

Previous work demonstrated that liposomes, containing an amino acid sequence that binds to hepatic heparan sulfate glycosaminoglycan, show effective targeting to liver hepatocytes. These liposomes were tested to determine whether they can deliver doxorubicin selectively to liver and hepatocytes in vivo. Fluid-phase liposomes contained a lipid-anchored 19-amino acid glycosaminoglycan targeting peptide. Liposomes were loaded with doxorubicin and were non-leaky in the presence of serum. After intravenous administration to mice, organs were harvested and the doxorubicin content extracted and measured by fluorescence intensity and by fluorescence microscopy. The liposomal doxorubicin was recovered almost entirely from liver, with only trace amounts detectable in heart, lung, and kidney. Fluorescence microscopy demonstrated doxorubicin preferentially in hepatocytes, also in non-parenchymal cells of the liver, but not in cells of heart, lung or kidney. The doxorubicin was localized within liver cell nuclei within 5 min after intravenous injection. These studies demonstrated that liposomal doxorubicin can be effectively delivered to hepatocytes by targeting the heparan sulfate glycosaminoglycan of liver tissue. With the composition described here, the doxorubicin was rapidly released from the liposomes without the need for an externally supplied stimulus.

Cellular organization of normal mouse liver: a histological, quantitative immunocytochemical, and fine structural analysis.

The cellular organization of normal mouse liver was studied using light and electron microscopy and quantitative immunocytochemical techniques. The general histological organization of the mouse liver is similar to livers of other mammalian species, with a lobular organization based on the distributions of portal areas and central venules. The parenchymal hepatocytes were detected with immunocytochemical techniques to recognize albumin or biotin containing cells. The macrophage Kupffer cells were identified with F4-80 immunocytochemistry, Ito stellate cells were identified with GFAP immunocytochemistry, and endothelial cells were labeled with the CD-34 antibody. Kupffer cells were labeled with intravascularly administered fluorescently labeled latex microspheres of both large (0.5 mum) and small (0.03 mum) diameters, while endothelial cells were labeled only with small diameter microspheres. Neither hepatocytes nor Ito stellate cells were labeled by intravascularly administered latex microspheres. The principal fine structural features of hepatocytes and non-parenchymal cells of mouse liver are similar to those reported for rat. Counts of immunocytochemically labeled cells with stained nuclei indicated that hepatocytes constituted approximately 52% of all labeled cells, Kupffer cells about 18%, Ito cells about 8%, and endothelial cells about 22% of all labeled cells. Approximately, 35% of the hepatocytes contained two nuclei; none of the Kupffer or Ito cells were double nucleated. The presence of canaliculi and a bile duct system appear similar to that reported for other species. The cellular organization of the mouse liver is quite similar to that of other mammalian species, confirming that the mouse presents a useful animal model for studies of liver structure and function.

Liposomal polyethyleneglycol and polyethyleneglycol-peptide combinations for active targeting to liver in vivo.

This report describes the development and evaluation of a range of polyethyleneglycol and polyethyleneglycol-peptide liposome formulations that effectively target liver in vivo. A 19-amino-acid sequence from the N-terminal region of the circumsporozoite protein of Plasmodium berghei was attached to the distal end of di22:1-aminopropane-polyethyleneglycol(3400), and incorporated into liposomes containing di22:1-phosphatidylcholine and di22:1-phosphatidylethanolamine-polyethyleneglycol(5000). By systematically varying the mole fractions of both the lipid-polyethyleneglycol and the lipid-polyethyleneglycol-peptide conjugates, and screening for serum-induced aggregation in vitro, a serum-stable range of formulations was established. These stable formulations were tested for binding to Hepa 1-6 liver cells in culture, and from these results three formulations were prepared for intravenous administration in mice. All three formulations exhibited effective liposome targeting to the liver, with approximately 80% of the total injected dose recovered in the liver within 15 min. Uptake by liver cells was more than 600-fold higher than uptake by those in the heart, and more than 200-fold higher than uptake by lung or kidney cells. Effective targeting to liver in vivo was successful after repeated (up to three) administrations to the host at 14-day intervals. All formulations prepared for in vivo administration were stable in the presence of serum, as measured by complete retention of entrapped calcein dye. The formulation with the lowest mole fractions of peptide and polyethyleneglycol was the most cost-effective in terms of encapsulation efficiency and minimal use of peptide and polymer compounds. The in vitro biophysical screening, followed by cell culture testing, reduced the number of animals required to develop an effective set of targeted liposome formulations for in vivo application.

Liposomes incorporating a Plasmodium amino acid sequence target heparan sulfate binding sites in liver.

Previous studies demonstrated that intravenously administered liposomes, incorporating a peptide from the Plasmodium circumsporozoite protein, accumulate rapidly and selectively in mouse liver. The present investigation was designed to determine the molecular components in liver responsible for liposome targeting. Studies of liver tissue slices demonstrated that immunoreactivity for heparan sulfate proteoglycan (HSPG), but not other tested proteoglycans, was distributed along sinusoidal borders of liver; this immunoreactivity appeared associated with nonparenchymal cells of the sinusoids and with the basolateral portion of hepatocytes. Peptide-containing liposomes bound to liver tissue in a pattern similar to the distribution of heparan sulfate immunoreactivity, either after intravenous injection of liposomes in vivo or after incubation of liposomes with liver slices in vitro. Control liposomes, without the peptide, displayed very light binding without a pattern. Pretreatment of liver slices with heparinase, but not chondroitinase or hyaluronidase, eliminated peptide-containing liposome binding, but did not affect binding of control liposomes. Coincubation of peptide-containing liposomes with heparin, but not with other glycosaminoglycans, markedly inhibited liposome binding to liver slices. N-desulfated and O-desulfated heparins individually were less effective inhibitors of liposome binding than was heparin. These results indicate that liposomes containing a peptide from Plasmodium target liver tissue by binding to HSPGs in the extracellular matrix.

Repair of spinal cord transection and its effects on muscle mass and myosin heavy chain isoform phenotype.

A number of significant advances have been developed for treating spinal cord injury during the past two decades. The combination of peripheral nerve grafts and acidic fibroblast growth factor (hereafter referred to as PNG) has been shown to partially restore hindlimb function. However, very little is known about the effects of such treatments in restoring normal muscle phenotype. The primary goal of the current study was to test the hypothesis that PNG would completely or partially restore 1) muscle mass and muscle fiber cross-sectional area and 2) the slow myosin heavy chain phenotype of the soleus muscle. To test this hypothesis, we assigned female Sprague-Dawley rats to three groups: 1) sham control, 2) spinal cord transection (Tx), and 3) spinal cord transection plus PNG (Tx+PNG). Six months following spinal cord transection, the open-field test was performed to assess locomotor function, and then the soleus muscles were harvested and analyzed. SDS-PAGE for single muscle fiber was used to evaluate the myosin heavy chain (MHC) isoform expression pattern following the injury and treatment. Immunohistochemistry was used to identify serotonin (5-HT) fibers in the spinal cord. Compared with the Tx group, the Tx+PNG group showed 1) significantly improved Basso, Beattie, and Bresnahan scores (hindlimb locomotion test), 2) less muscle atrophy, 3) a higher percentage of slow type I fibers, and 4) 5-HT fibers distal to the lesion site. We conclude that the combined treatment of PNG is partially effective in restoring the muscle mass and slow phenotype of the soleus muscle in a T-8 spinal cord-transected rat model.

Prenatal manganese levels linked to childhood behavioral disinhibition.

Although manganese (Mn) is an essential mineral, high concentrations of the metal can result in a neurotoxic syndrome affecting dopamine balance and behavior control. We report an exploratory study showing an association between Mn deposits in tooth enamel, dating to the 20th and 62-64th gestational weeks, and childhood behavioral outcomes. In a sample of 27 children, 20th week Mn level was significantly and positively correlated with measures of behavioral disinhibition, specifically, play with a forbidden toy (36 months), impulsive errors on a continuous performance and a children's Stroop test (54 months), parents' and teachers' ratings of externalizing and attention problems on the Child Behavior Checklist (1st and 3rd grades), and teacher ratings on the Disruptive Behavior Disorders Scale (3rd grade). By way of contrast, Mn level in tooth enamel formed at the 62-64th gestational week was correlated only with teachers' reports of externalizing behavior in 1st and 3rd grades. Although the source(s) of Mn exposure in this sample are unknown, one hypothesis, overabsorption of Mn secondary to gestational iron-deficiency anemia, is discussed.

Effective targeting of liposomes to liver and hepatocytes in vivo by incorporation of a Plasmodium amino acid sequence.

PURPOSE: Several species of the protozoan Plasmodium effectively target mammalian liver during the initial phase of host invasion. The purpose of this study was to demonstrate that a Plasmodium targeting amino acid sequence can be engineered into therapeutic nanoparticle delivery systems. METHODS: A 19-amino peptide from the circumsporozoite protein of Plasmodium berghei was prepared containing the conserved region I as well as a consensus heparan sulfate proteoglycan binding sequence. This peptide was attached to the distal end of a lipid-polyethylene glycol bioconjugate. The bioconjugate was incorporated into phosphatidylcholine liposomes containing fluorescently labeled lipids to follow blood clearance and organ distribution in vivo. RESULTS: When administered intravenously into mice, the peptide-containing liposomes were rapidly cleared from the circulation and were recovered almost entirely in the liver. Fluorescence and electron microscopy demonstrated that the liposomes were accumulated both by nonparenchymal cells and hepatocytes, with the majority of the liposomal material associated with hepatocytes. Accumulation of liposomes in the liver was several hundredfold higher compared to heart, lung, and kidney, and more than 10-fold higher compared to spleen. In liver slice experiments, liposome binding was specific to sites sensitive to heparinase. CONCLUSIONS: Incorporation of amino acid sequences that recognize glycosaminoglycans is an effective strategy for the development of targeted drug delivery systems.

Re-growth of catecholaminergic fibers and protection of cholinergic spinal cord neurons in spinal repaired rats.

The extent of re-growth of catecholaminergic fibers, the survival of cholinergic neurons and the degree of autonomic dysreflexia were assessed in complete spinal cord-transected adult rats that received a repair treatment of peripheral nerve grafts and acidic fibroblast growth factor (aFGF). The rats were randomly divided into three groups: (1) sham control group (laminectomy only); (2) spinal cord transection at T8 (transected group); and (3) spinal cord transection at T8, followed by aFGF treatment and peripheral nerve graft (repaired group). The spinal cords and brains of all rats were collected at 6 months post-surgery. Immunohistochemistry for tyrosine hydroxylase (TH) and dopamine-beta-hydroxylase (DBH), and fluoro-gold (FG) retrograde tracing were used to evaluate axon growth across the damage site, and immunocytochemistry for choline acetyl transferase (ChAT) was used to evaluate cholinergic neuronal cell survival following the injury and treatment. When comparing with the transected group, the repaired group showed: (1) lower elevation of mean arterial pressure during colorectal distension; (2) retrogradely labeled neurons in the hypothalamus, zona incerta, subcoeruleus nuclei and rostral ventrolateral medulla following application of FG below the repair site; (3) the presence of TH- and DBH-labeled axons below the lesion site; (4) higher numbers of ChAT-positive neurons in ventral horn and intermediolateral column near the lesion site. We conclude that peripheral nerve graft and aFGF treatments facilitate the re-growth of catecholaminergic fibers, also protect sympathetic preganglionic neurons and spinal motor neurons, and reduce autonomic dysfunction in a T-8 spinal cord-transected rat model.

Axon reactive B cells clonally expanded in the cerebrospinal fluid of patients with multiple sclerosis.

Demyelination and axonal loss have been described as the histological hallmarks of inflammatory lesions of multiple sclerosis (MS) and are the pathological correlates of persistent disability. However, the immune mechanisms underlying axonal damage in MS remain unknown. Here, we report the use of single chain-variable domain fragments (scFv) from clonally expanded cerebrospinal fluid (CSF) B cells to show the role of an anti-axon immune response in the central nervous system (CNS) in MS. The cellular and subcellular distribution of the antigen(s) recognized by these CSF-derived clonal scFv antibodies (CSFC-scFv Abs) was studied by immunochemical staining of brain tissues obtained at autopsy from patients with MS. Immunochemistry showed specific binding of CSFC-scFv Abs to axons in acute MS lesions. The stained axons showed three major types of axonal pathological changes: 1) linear axons, axonal ovoid formation, and axonal transection were seen in the myelinated white matter adjacent to the lesion; 2) accumulation of axonal ovoid formations and Wallerian degeneration were seen at the border between demyelinated lesions and the adjacent white matter; and 3) Wallerian degeneration occurred at the center and edge of acute demyelinated lesions. These findings suggest a B cell axonal specific immune response in the CNS in MS.

Patterns of afferent projections to the dentate gyrus studied in organotypic co-cultures.

Cholinergic axons originating from the septum form a characteristic layer of preterminal axons and apparent termination in the molecular layer of the hippocampal dentate gyrus. The present study explored the specificity of this characteristic axonal pattern, through the use of organotypic slice co-cultures. Slices of hippocampus were co-cultured with a slice from one of a variety of other potential sources of afferents, and the afferent axons were labeled histochemically or immunocytochemically to determine which afferents distribute within the dentate molecular layer in a pattern similar to that formed by septal cholinergic projections. Acetylcholinesterase (AChE) histochemistry demonstrated that cholinergic axons from septum, substantia innominata, and striatum all consistently targeted the inner molecular layer of the dentate gyrus. AChE-labeled cholinergic axons from dorsal lateral pontine tegmentum and from spinal cord sometimes formed this pattern, while axons from the habenula failed to extend into the dentate gyrus. Immunocytochemically identified monoaminergic axons from the substantia nigra, locus coeruleus, and raphe extended into co-cultured hippocampus; each of these afferent systems displayed a prominent axonal plexus within the hilus of the dentate, but only the raphe axons projected prominently to the molecular layer. These data demonstrate that the molecular layer of the dentate gyrus provides an attractive target zone for some cholinergic and monoaminergic afferents, but not all. Commonalities between neuronal populations that preferentially project to the molecular layer in vitro may offer clues regarding the axon guidance mechanisms that normally direct cholinergic axons to target sites in the dentate gyrus molecular layer.

Motor recovery and anatomical evidence of axonal regrowth in spinal cord-repaired adult rats.

Behavioral assessments of hindlimb motor recovery and anatomical assessments of extended axons of long spinal tracts were conducted in adult rats following complete spinal cord transection. Rats were randomly divided into 3 groups: 1) sham control group (laminectomy only; n = 12); 2) transection-only group, spinal cord transection at T8 (n = 20); and 3) experimental treatment group, spinal cord transection at T8, with peripheral nerve grafts (PNG) and application of acidic fibroblast growth factor (aFGF) (n = 14). The locomotor behavior and stepping of all rats were analyzed over a 6-month survival time using the Basso, Beattie, Bresnahan (BBB) open field locomotor test and the contact placing test. Immunohistochemistry for serotonin (5-HT), anterograde tracing with biotinylated dextran amine (BDA), and retrograde tracing with fluoro-gold were used to evaluate the presence of axons below the damage site following treatment. When compared with the transection-only group, the nerve graft with the aFGF group showed 1) significant improvement in hindlimb locomotion and stepping, 2) the presence of 5-HT-labeled axons below the lesion site at lumbar cord level (these were interpreted as regenerated axons from the raphe nuclei), 3) the presence of anterograde BDA labeling of corticospinal tract axons at the graft site and below, and 4) fluoro-gold retrograde labeling of neuron populations in motor cortex and in red nucleus, reticulospinal nuclei, raphe nuclei, and vestibular nuclei. We conclude that peripheral nerve grafts and aFGF treatments facilitate the regrowth of the spinal axons and improve hindlimb function in a T-8 spinal cord-transected rat model.

Assessment of factors regulating axon growth between the cortex and spinal cord in organotypic co-cultures: effects of age and neurotrophic factors.

Axon growth failure in the central nervous system (CNS) of adult animals is thought to be attributable to several factors, including an inadequate intrinsic growth response, the presence of inhibitory molecules, and a lack of adequate neurotrophic support. Here we use a new in vitro assay system to quantitatively assess growth of axons in cortex/spinal cord organotypic co-cultures from neonatal rats. Co-cultures of cortex and spinal cord were prepared from neonatal rats at P3 or P7, and by pairing cortex and spinal cords from different ages. Axon growth from the cortex to the spinal cord was assessed using DiI tract tracing techniques. Axons could be traced from the cortex to the spinal cord in co-cultures in which both tissues were obtained from P3 animals, whereas few axons crossed the cortex/spinal cord boundary in co-cultures from P7 animals. A larger number of axons could be traced across the boundary in co-cultures from P3 animals that were treated with neurotrophins (NGF, BDNF, or NT3), whereas neurotrophins produced minimal growth enhancement in P7 co-cultures. In mixed age co-cultures of P7 cortex with P3 spinal cord, moderate numbers of axons extended between the cortex and spinal cord when cultures were treated with neurotrophins, but few if any crossing axons were detected in co-cultures of P3 cortex with P7 spinal cords. These results indicate that successful growth of axons from the cortex to the spinal cord depends on the developmental age of the tissue terrain (the spinal cord and/or the interface between cortex and spinal cord explants), and to a lesser extent on the developmental state of the cortical neurons, and that axon growth between cortex and spinal cord can be enhanced by exogenous neurotrophins. These co-cultures provide a potentially useful assay for factors that affect axon growth that is intermediate between assays based on dissociated neurons and the intact tissue terrain.

Absence of selectivity in the loss of neurons from the developing cortical subplate of the rat.

Neurons of the cortical subplate display evidence of cell death, although a significant population survives to the mature brain. The present study examined different populations of neurons to determine if the loss of cells was specific for a particular cell type. Immunocytochemical procedures for neurons expressing GluR2/3, GAD, or NPY, were used on tissue sections taken from animals at gestational day 18 to postnatal day 21. The rate of loss of labeled cells was similar for all groups of neurons. Thus, these data reveal no evidence that the loss of subplate neurons is specific to any major cell type.