Diagnosis of traumatic brain injury using miRNA signatures in nanomagnetically isolated brain-derived extracellular vesicles.

The accurate diagnosis and clinical management of traumatic brain injury (TBI) is currently limited by the lack of accessible molecular biomarkers that reflect the pathophysiology of this heterogeneous disease. To address this challenge, we developed a microchip diagnostic that can characterize TBI more comprehensively using the RNA found in brain-derived extracellular vesicles (EVs). Our approach measures a panel of EV miRNAs, processed with machine learning algorithms to capture the state of the injured and recovering brain. Our diagnostic combines surface marker-specific nanomagnetic isolation of brain-derived EVs, biomarker discovery using RNA sequencing, and machine learning processing of the EV miRNA cargo to minimally invasively measure the state of TBI. We achieved an accuracy of 99% identifying the signature of injured vs. sham control mice using an independent blinded test set (N = 77), where the injured group consists of heterogeneous populations (injury intensity, elapsed time since injury) to model the variability present in clinical samples. Moreover, we successfully predicted the intensity of the injury, the elapsed time since injury, and the presence of a prior injury using independent blinded test sets (N = 82). We demonstrated the translatability in a blinded test set by identifying TBI patients from healthy controls (AUC = 0.9, N = 60). This approach, which can detect signatures of injury that persist across a variety of injury types and individual responses to injury, more accurately reflects the heterogeneity of human TBI injury and recovery than conventional diagnostics, opening new opportunities to improve treatment of traumatic brain injuries.

DLG-1 is a MAGUK similar to SAP97 and is required for adherens junction formation.

Cellular junctions are critical for intercellular communication and for the assembly of cells into tissues. Cell junctions often consist of tight junctions, which form a permeability barrier and prevent the diffusion of lipids and proteins between cell compartments, and adherens junctions, which control the adhesion of cells and link cortical actin filaments to attachment sites on the plasma membrane. Proper tight junction formation and cell polarity require the function of membrane-associated guanylate kinases (MAGUKs) that contain the PDZ protein-protein interaction domain. In contrast, less is known about how adherens junctions are assembled. Here we describe how the PDZ-containing protein DLG-1 is required for the proper formation and function of adherens junctions in Caenorhabditis elegans. DLG-1 is a MAGUK protein that is most similar in sequence to mammalian SAP97, which is found at both synapses of the CNS, as well as at cell junctions of epithelia. DLG-1 is localized to adherens junctions, and DLG-1 localization is mediated by an amino-terminal domain shared with SAP97 but not found in other MAGUK family members. DLG-1 recruits other proteins and signaling molecules to adherens junctions, while embryos that lack DLG-1 fail to recruit the proteins AJM-1 and CPI-1 to adherens junctions. DLG-1 is required for the proper organization of the actin cytoskeleton and for the morphological elongation of embryos. In contrast to other proteins that have been observed to affect adherens junction assembly and function, DLG-1 is not required to maintain cell polarity. Our results suggest a new function for MAGUK proteins distinct from their role in cell polarity.

Cooperative regulation of AJM-1 controls junctional integrity in Caenorhabditis elegans epithelia.

The function of epithelial cell sheets depends on the integrity of specialized cell-cell junctions that connect neighbouring cells. We have characterized the novel coiled-coil protein AJM-1, which localizes to an apical junctional domain of Caenorhabditis elegans epithelia basal to the HMR-HMP (cadherin-catenin) complex. In the absence of AJM-1, the integrity of this domain is compromised. Proper AJM-1 localization requires LET-413 and DLG-1, homologues of the Drosophila tumour suppressors Scribble and Discs large, respectively. DLG-1 physically interacts with AJM-1 and is required for its normal apical distribution, and LET-413 mediates the rapid accumulation of both DLG-1 and AJM-1 in the apical domain. In the absence of both dlg-1 and let-413 function AJM-1 is almost completely lost from apical junctions in embryos, whereas HMP-1 (alpha-catenin) localization is only mildly affected. We conclude that LET-413 and DLG-1 cooperatively control AJM-1 localization and that AJM-1 controls the integrity of a distinct apical junctional domain in C. elegans.

Binding of neuronal nitric-oxide synthase (nNOS) to carboxyl-terminal-binding protein (CtBP) changes the localization of CtBP from the nucleus to the cytosol: a novel function for targeting by the PDZ domain of nNOS.

Recent work suggests a role for PDZ domains in the targeting of binding partners to specific sites in the cell. To identify whether the PDZ domain of neuronal nitric-oxide synthase (nNOS) can play such a role, we performed affinity chromatography of brain extract with the nNOS PDZ domain. We identified the carboxyl-terminal-binding protein (CtBP), a phosphoprotein first identified as a binding partner to adenovirus E1A, as a nNOS binding partner. CtBP interacts with the PDZ domain of nNOS, and this interaction can be competed with peptide that binds to the PDZ peptide-binding site. In addition, binding of CtBP to nNOS is dependent on its carboxyl-terminal sequence -DXL, residues conserved between species that fit the canonical sequence for nNOS PDZ binding. Immunoprecipitation studies show that CtBP and nNOS associate in the brain. When CtBP is expressed in Madin-Darby canine kidney cells, its distribution is primarily nuclear; however, when CtBP is co-expressed with nNOS, its localization becomes more cytosolic. This change in CtBP localization does not occur when its carboxyl-terminal nNOS PDZ binding motif is mutated or when CtBP is co-expressed with postsynaptic density 95, another PDZ domain-containing protein. Taken together, our data suggest a new function for nNOS as a regulator of CtBP nuclear localization.

P2Y receptors of MDCK cells: epithelial cell regulation by extracellular nucleotides.

1. Madin-Darby canine kidney (MDCK) cells, a well- differentiated renal epithelial cell line derived from distal tubule/collecting duct, respond to extracellular nucleotides by altering ion flux and the production of arachidonic acid-derived products, in particular prostaglandin E2 (PGE2). Our work has defined the receptors and signalling events involved in such responses. 2. We have found evidence for expression of at least three P2Y receptor subtypes (P2Y1, P2Y2 and P2Y11) in MDCK-D1 cells, a subclone from parental MDCK. 3. These receptors appear to couple to increases in calcium and protein kinase C activity, probably via a Gq/G11-mediated activation of phospholipase C. 4. In addition, P2Y receptor activation can promote a prominent increase in cAMP. This includes both a P2Y2 receptor-mediated cyclo-oxygenase (COX)-dependent component and another COX-independent component mediated by other P2Y receptors. 5. We have documented that changing media in which cells are grown releases ATP and, in turn, activates P2Y receptors. Such release of ATP contributes in a major way to basal cAMP levels in these cells. 6. The data indicate that MDCK cells are a useful model to define the regulation of epithelial cells by extracellular nucleotides. Of particular note, spontaneous or stretch-induced release of ATP and subsequent activation of one or more P2Y receptors contributes to establishing the basal activity of signalling pathways.

Postsynaptic targeting of MAGUKs mediated by distinct N-terminal domains.

Postsynaptic targeting of PSD-95 has been extensively studied; however, little is known about how other MAGUKs are localized. Proper targeting of PSD-95 requires dual palmitoylation of an N-terminal motif. We now find that the N-termini of closely related PSD-93 and SAP-102 are also involved in postsynaptic targeting. PSD-93 is N-terminally palmitoylated; however, unlike PSD-95, palmitoylation does not explain the necessity of the N-terminus for PSD-93 postsynaptic targeting. Furthermore, when the N-terminus of PSD-95 is replaced with the first 30 or 64, but not the first 10, amino acids of PSD-93, the chimera is targeted to postsynaptic sites independent of palmitoylation. Similarly, when the N-terminus of PSD-95 is replaced with the non-palmitoylated N-terminus of SAP-102, postsynaptic targeting is maintained. These results suggest that MAGUKs contain diverse signals within their N-termini for postsynaptic targeting.

Nitric oxide mediated induction of cytochrome c oxidase mRNA and protein in a mouse macrophage cell line.

Neurons that express neuronal nitric oxide synthase (nNOS) are selectively spared from nitric oxide (NO)-induced cytotoxicity in acute cerebral ischemia and neurodegenerative conditions but the mechanism of this resistance is unknown. To identify specific gene products which may mediate this resistance, we performed polymerase chain reaction (PCR)-based subtractive hybridization on a mouse macrophage cell line treated with either L-NG-nitroarginine methyl ester (L-NAME, 1 mM, 1 h), an inhibitor of NOS, or with diethylamine NONOate (DEA NONO, 200 microM, 1 h), an NO donor. NO-treated cultures showed an acute induction of mRNA (less than 1 h after treatment) and protein (15 min) for the mitochondrial enzyme cytochrome c oxidase (CcO) as shown by Northern or Western blot analysis, respectively. Cytochrome c oxidase activity assay showed constant activity in NO-treated cultures, as compared to L-NAME-treated cultures. NO directly inhibits CcO, the terminal electron acceptor in mitochondrial oxidative respiration. Up-regulation of this enzyme by NO, therefore, appears to maintain vital CcO activity and cellular energy stores, thus contributing to selective sparing of nNOS neurons.

Ion channel clustering by membrane-associated guanylate kinases. Differential regulation by N-terminal lipid and metal binding motifs.

The postsynaptic density protein PSD-95 and related membrane-associated guanylate kinase (MAGUK) proteins assemble signal transduction complexes at sites of cell-cell contact including synapses. Whereas PSD-95 and PSD-93 occur only at postsynaptic sites in hippocampal neurons, SAP-102 also occurs in axons. In heterologous cells, PSD-95 and PSD-93 mediate cell surface ion channel clustering, but SAP-102 and SAP-97 do not. This selective ion channel clustering activity by MAGUKs is explained by differential palmitoylation, as PSD-93 and PSD-95 are palmitoylated though SAP-97, and SAP-102 are not. Rather than being palmitoylated, we find that N-terminal cysteines from SAP-102 tightly bind to zinc. And, appending the N terminus of SAP-102 to PSD-95 results in localization of the chimera to both axons and dendrites. These data suggest that lipid modifications and heavy metal associations with the N termini of MAGUKs mediate differential functions and subcellular localizations of these synaptic scaffolds.

Dual palmitoylation of PSD-95 mediates its vesiculotubular sorting, postsynaptic targeting, and ion channel clustering.

Postsynaptic density-95 (PSD-95/SAP-90) is a palmitoylated peripheral membrane protein that scaffolds ion channels at excitatory synapses. To elucidate mechanisms for postsynaptic ion channel clustering, we analyzed the cellular trafficking of PSD-95. We find that PSD-95 transiently associates with a perinuclear membranous compartment and traffics with vesiculotubular structures, which migrate in a microtubule-dependent manner. Trafficking of PSD-95 with these vesiculotubular structures requires dual palmitoylation, which is specified by five consecutive hydrophobic residues at the NH(2) terminus. Mutations that disrupt dual palmitoylation of PSD-95 block both ion channel clustering by PSD-95 and its synaptic targeting. Replacing the palmitoylated NH(2) terminus of PSD-95 with alternative palmitoylation motifs at either the NH(2) or COOH termini restores ion channel clustering also induces postsynaptic targeting, respectively. In brain, we find that PSD-95 occurs not only at PSDs but also in association with intracellular smooth tubular structures in dendrites and spines. These data imply that PSD-95 is an itinerant vesicular protein; initial targeting of PSD-95 to an intracellular membrane compartment may participate in postsynaptic ion channel clustering by PSD-95.

Cypin: a cytosolic regulator of PSD-95 postsynaptic targeting.

Postsynaptic density 95 (PSD-95/SAP-90) is a membrane associated guanylate kinase (GK) PDZ protein that scaffolds glutamate receptors and associated signaling networks at excitatory synapses. Affinity chromatography identifies cypin as a major PSD-95-binding protein in brain extracts. Cypin is homologous to a family of hydrolytic bacterial enzymes and shares some similarity with collapsin response mediator protein (CRMP), a cytoplasmic mediator of semaphorin III signalling. Cypin is discretely expressed in neurons and is polarized to basal membranes in intestinal epithelial cells. Overexpression of cypin in hippocampal neurons specifically perturbs postsynaptic trafficking of PSD-95 and SAP-102, an effect not produced by overexpression of other PDZ ligands. In fact, PSD-95 can induce postsynaptic clustering of an otherwise diffusely localized K+ channel, Kv1.4. By regulating postsynaptic protein sorting, cypin may influence synaptic development and plasticity.

Interaction of neuronal nitric-oxide synthase and phosphofructokinase-M.

Neurons that express neuronal nitric-oxide synthase (nNOS) are resistant to NO-induced neurotoxicity; however, the mechanism by which these neurons are protected is not clear. To identify proteins possibly involved in this process, we performed affinity chromatography with the nNOS PDZ domain, a N-terminal motif that mediates protein interactions. Using this method to fractionate soluble tissue extracts, we identified the muscle isoform of phosphofructokinase (PFK-M) as a protein that binds to nNOS both in brain and skeletal muscle. PFK-M interacts with the PDZ domain of nNOS, and nNOS-PFK-M binding can be competed by peptides that bind to the PDZ domain of nNOS. We found that nNOS is significantly associated with PFK-M in skeletal muscle because nNOS can be immunodepleted from cytosolic skeletal muscle extracts using an antibody directed against PFK-M. In brain, nNOS and PFK-M are both enriched in synaptosomes, and specifically, in the synaptic vesicle fraction, where they can interact. At the cellular level, PFK-M is enriched in neurons that express nNOS protein. As fructose-1, 6-bisphosphate, the product of PFK activity, is neuroprotective, the interaction of nNOS and PFK may contribute to neuroprotection of nNOS positive cells.

Regulation of sensory neuron precursor proliferation by cyclic GMP-dependent protein kinase.

Cyclic GMP (cGMP) is a molecular messenger involved in diverse cellular processes. Recently, cGMP-dependent protein kinase (cGK) type II was determined to be a regulator of endochondral ossification and bone growth, identifying a role for cGMP in the regulation of cellular proliferation. Here, we demonstrate the presence of cGK type I (cGKI) in cells of the developing trigeminal ganglia. cGKI occurs in some proliferating precursors as evidenced by double labeling with an antibody to cGKI and 5-bromo-2'-deoxyuridine(BrdU) incorporation. Inhibition of cGKI with KT5823 or Rp-8-(4-chlorophenylthio)-guanosine-3',5'-cyclic monophosphorothioate (Rp-8-pCPT-cGMPS) in chick embryos results in a 30-40% decrease in trigeminal ganglia cell number, and this effect is independent of nitric oxide synthase (NOS). In addition, inhibition of cGKI with Rp-8-pCPT-cGMPS results in a 60% decrease in BrdU incorporation in the trigeminal ganglia of embryonic day 5 chicks. We find that PC12 cells expressing cGKI proliferate more rapidly and incorporate more BrdU than do control cells. The cGKI inhibitor Rp-8-pCPT-cGMPS decreases proliferation and BrdU incorporation in transfected PC12 cells but has no effect on control cells. The PC12 cells do not express NOS, indicating that this effect is also independent of NOS. Thus, cGKI regulates the proliferation of sensory neurons as a result of activation of a NOS-independent pathway, representing a novel pathway by which the number of sensory neurons is regulated.

PDZ proteins bind, cluster, and synaptically colocalize with Eph receptors and their ephrin ligands.

Localizing cell surface receptors to specific subcellular positions can be critical for their proper functioning, as most notably demonstrated at neuronal synapses. PDZ proteins apparently play critical roles in such protein localizations. Receptor tyrosine kinases have not been previously shown to interact with PDZ proteins in vertebrates. We report that Eph receptors and their membrane-linked ligands all contain PDZ recognition motifs and can bind and be clustered by PDZ proteins. In addition, we find that Eph receptors and ligands colocalize with PDZ proteins at synapses. Thus, PDZ proteins may play critical roles in localizing vertebrate receptor tyrosine kinases and/or their ligands and may be particularly important for Eph function in guidance or patterning or at the synapse.

Dual role of protein kinase C in the regulation of cPLA2-mediated arachidonic acid release by P2U receptors in MDCK-D1 cells: involvement of MAP kinase-dependent and -independent pathways.

Defining the mechanism for regulation of arachidonic acid (AA) release is important for understanding cellular production of AA metabolites, such as prostaglandins and leukotrienes. Here we have investigated the differential roles of protein kinase C (PKC) and mitogen-activated protein (MAP) kinase in the regulation of cytosolic phospholipase A2 (cPLA2)-mediated AA release by P2U-purinergic receptors in MDCK-D1 cells. Treatment of cells with the P2U receptor agonists ATP and UTP increased PLA2 activity in subsequently prepared cell lysates. PLA2 activity was inhibited by the cPLA2 inhibitor AACOCF3, as was AA release in intact cells. Increased PLA2 activity was recovered in anti-cPLA2 immunoprecipitates of lysates derived from nucleotide-treated cells, and was lost from the immunodepleted lysates. Thus, cPLA2 is responsible for AA release by P2U receptors in MDCK-D1 cells. P2U receptors also activated MAP kinase. This activation was PKC-dependent since phorbol 12-myristate 13-acetate (PMA) promoted down-regulation of PKC-eliminated MAP kinase activation by ATP or UTP. Treatment of cells with the MAP kinase cascade inhibitor PD098059, the PKC inhibitor GF109203X, or down-regulation of PKC by PMA treatment, all suppressed AA release promoted by ATP or UTP, suggesting that both MAP kinase and PKC are involved in the regulation of cPLA2 by P2U receptors. Differential effects of GF109203X on cPLA2-mediated AA release and MAP kinase activation, however, were observed: at low concentrations, GF109203X inhibited AA release promoted by ATP, UTP, or PMA without affecting MAP kinase activation. Since GF109203X is more selective for PKCalpha, PKCalpha may act independently of MAP kinase to regulate cPLA2 in MDCK-D1 cells. This conclusion is further supported by data showing that PMA-promoted AA release, but not MAP kinase activation, was suppressed in cells in which PKCalpha expression was decreased by antisense transfection. Based on these data, we propose a model whereby both MAP kinase and PKC are required for cPLA2-mediated AA release by P2U receptors in MDCK-D1 cells. PKC plays a dual role in this process through the utilization of different isoforms: PKCalpha regulates cPLA2-mediated AA release independently of MAP kinase, while other PKC isoforms act through MAP kinase activation. This model contrasts with our recently demonstrated mechanism (J. Clin. Invest. 99:1302-1310.) whereby alpha1-adrenergic receptors in the same cell type regulate cPLA2-mediated AA release only through sequential activation of PKC and MAP kinase.

P2-purinoceptors utilize multiple signalling pathways in MDCK-D1 cells.

1. Madin-Darby canine kidney (MDCK) cells are a widely used model system for the study of epithelial cells. We have utilized a clonal variant, MDCK-D1, to examine signalling by P2-purinoceptors. 2. Several lines of evidence that lead us to conclude that MDCK-D1 cells co-express P2a- and P2y-purinoceptors and that both subtypes are linked to the release of arachidonic acid and metabolites (AA) include: (a) relative potency of nucleotide analogues in promoting AA release; (b) blockade by the antagonist suramin of response to the P2Y-selective agonist, 2-methylthio ATP (2-MT-ATP), but not to the P2a-selective agonist, UTP; and (c) additivity of response to 2-MT-ATP and UTP. AA release is a consequence of activation of phospholipase A2 (PLA2), most likely the 85 kDa cytosolic PLA2. 3. Treatment of MDCK-D1 cells with ATP, but not UTP, increases inositol 1,4,5-trisphosphate formation while both UTP and ATP increase phosphatidylcholine hydrolysis, ATP, UTP, and 2-MT-ATP can also stimulate phospholipase D activity. 4. Purine nucleotides increase cellular cAMP levels in MDCK-D1 cells in a manner that depends, at least in part, on activation of cyclooxygenase, since cAMP generation stimulated by ATP or UTP is inhibited by treatment of cells with indomethacin. Because cyclooxygenase-derived PGE2 can bind to prostaglandin receptors and stimulate synthesis of cAMP, nucleotides may raise cAMP in an autocrine or paracrine fashion. 5. Taken together, these results indicate that MDCK-D1 cells co-express P2a and P2y-purinoceptors and that these receptors utilize several mechanisms to regulate cell function, including activation of multiple phospholipases and autocrine/paracrine action of products.

Heterogeneity of P2u- and P2y-purinergic receptor regulation of phospholipases in MDCK cells.

We have characterized the signaling pathways of purinergic receptors present on the renal epithelial cell line, Madin-Darby canine kidney (MDCK, D1 subclone). Several lines of evidence are consistent with the conclusion that coexisting P2u and P2y receptors release arachidonic acid and metabolites (AA) from MDCK-D1 cells: 1) relative potencies of nucleotide analogues, 2) blockade of P2y agonist- but not P2u agonist-mediated release by suramin, and 3) additivity by 2-methylthio-ATP and UTP. Differences exist between the signaling pathways of the two receptors: pertussis toxin treatment partially inhibits P2u- but not P2y-mediated AA release, and P2y (but not P2u) receptors appear to stimulate D-myo-inositol 1,4,5-trisphosphate production. P2u-receptor occupancy results in both homologous and heterologous desensitization; P2y-receptor occupancy elicits only homologous desensitization. Both receptors stimulate phosphatidylcholine hydrolysis via phospholipase C activation. However, AA release appears to result from phospholipid deacylation by phospholipase A2 activation, rather than from alternate pathways that may include PLC activation. These results demonstrate for the first time that two subtypes of P2-purinergic receptors, P2u and P2y receptors, coexist on a single renal epithelium cell type and that these two receptor subtypes can promote AA release, probably via activation of PLA2.

Protein kinase C alpha mediates phospholipase D activation by nucleotides and phorbol ester in Madin-Darby canine kidney cells. Stimulation of phospholipase D is independent of activation of polyphosphoinositide-specific phospholipase C and phospholipase A2.

Protein kinase C (PKC) has been implicated in the activation of phospholipase D (PLD) in a number of systems. By antisense technology, we have "knocked out" alpha and beta isoforms of PKC to study the role of these isoforms in PLD activation in Madin-Darby canine kidney (MDCK) cells. To this end, we have studied PLD activation by phorbol 12-myristate 13-acetate (PMA), ATP, UTP, and 2-methylthio-ATP in cells labeled with [3H]palmitic acid. [3H]Phosphatidylethanol (PEt) production catalyzed by PLD in the presence of ethanol was time- and concentration-dependent in PMA- and nucleotide-stimulated cells. In Ca(2+)-free medium, [3H]PEt accumulation was diminished for all stimuli assayed. Treatment of cells with chelerythrine, an inhibitor of PKC, and phorbol ester down-regulation of PKC inhibited [3H]PEt production by both PMA and nucleotides. In cells transfected with antisense PKC alpha or both PKC alpha and PKC beta, PLD activation was inhibited by both PMA and nucleotides, whereas in cells transfected with antisense PKC beta, PLD activation was similar to that of control cells. Moreover, inhibition of polyphosphoinositide-specific PLC (by neomycin) or of release of arachidonic acid and arachidonic acid metabolites (by nordihydroguaiaretic acid or by indomethacin) failed to decrease [3H]PEt accumulation in PMA- and nucleotide-stimulated MDCK-D1 cells. From these data, we conclude that in MDCK-D1 cells PMA and nucleotide receptors utilize PKC alpha to regulate PLD activity and that PLD activation is independent of the activation of polyphosphoinositide-specific PLC and phospholipase A2-mediated release of arachidonic acid or arachidonic acid metabolites.

Intracerebral grafting of cultured autologous skin fibroblasts into the rat striatum: an assessment of graft size and ultrastructure.

To identify a suitable donor cell population for gene therapy applications to the central nervous system, primary fibroblasts isolated from skin biopsies and maintained in culture are employed as autologous cells for intracerebral grafting within the adult rat striatum. Results from the present investigation reveal that cultured primary skin fibroblasts cease to proliferate once they reach confluence; these cells are thus contact inhibited in vitro. Following implantation within the striatum, the volume of the primary fibroblast grafts, stained immunohistochemically for fibronectin, does not differ significantly at 3 and 8 weeks. The graft size is dependent on the density of the cell suspension, but not dependent on either the number of passages the cells are taken through in culture prior to grafting or on the postoperative survival period. Ultrastructural evidence reveals that at 8 weeks the grafts are composed primarily of collagen and fibroblasts with rough endoplasmic reticulum and vesicles. Reactive astrocytic processes and phagocytic cells are also present in the grafts. The grafts are extensively vascularized with capillaries composed of nonfenestrated endothelium; intercellular junctions are evident at sites of apposition between endothelial cells. It is concluded that primary skin fibroblasts are able to survive for at least 8 weeks following intracerebral implantation and continue to synthesize collagen and fibronectin in vivo. Also, the grafts maintain a constant volume between 3 and 8 weeks, thereby indicating that primary skin fibroblasts do not produce tumors. Finally, dynamic host-to-graft interactions--including phagocytic migration, astrocytic hypertrophy and infiltration within the grafts, and angiogenesis--are features that constitute the structural integration of primary skin fibroblasts grafted within the adult rat central nervous system.