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.

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.

Kappa opioid control of seizures produced by a virus in an animal model.

Epilepsy remains a major medical problem of unknown aetiology. Potentially, viruses can be environmental triggers for development of seizures in genetically vulnerable individuals. An estimated half of encephalitis patients experience seizures and approximately 4% develop status epilepticus. Epilepsy vulnerability has been associated with a dynorphin promoter region polymorphism or low dynorphin expression genotype, in man. In animals, the dynorphin system in the hippocampus is known to regulate excitability. The present study was designed to test the hypothesis that reduced dynorphin expression in the dentate gyrus of hippocampus due to periadolescent virus exposure leads to epileptic responses. Encephalitis produced by the neurotropic Borna disease virus in the rat caused epileptic responses and dynorphin to disappear via dentate granule cell loss, failed neurogenesis and poor survival of new neurons. Kappa opioid (dynorphin) agonists prevented the behavioural and electroencephalographic seizures produced by convulsant compounds, and these effects were associated with an absence of dynorphin from the dentate gyrus granule cell layer and upregulation of enkephalin in CA1 interneurons, thus reproducing a neurochemical marker of epilepsy, namely low dynorphin tone. A key role for kappa opioids in anticonvulsant protection provides a framework for exploration of viral and other insults that increase seizure vulnerability and may provide insights into potential interventions for treatment of epilepsy.

A role for endocannabinoids in viral-induced dyskinetic and convulsive phenomena.

Dyskinesias and seizures are both medically refractory disorders for which cannabinoid-based treatments have shown early promise as primary or adjunctive therapy. Using the Borna disease (BD) virus rat, an animal model of viral encephalopathy with spontaneous hyperkinetic movements and seizure susceptibility, we identified a key role for endocannabinoids in the maintenance of a balanced tone of activity in extrapyramidal and limbic circuits. BD rats showed significant elevations of the endocannabinoid anandamide in subthalamic nucleus, a relay nucleus compromised in hyperkinetic disorders. While direct and indirect cannabinoid agonists had limited motor effects in BD rats, abrupt reductions of endocannabinoid tone by the CB1 antagonist SR141716A (0.3 mg/kg, i.p.) caused seizures characterized by myoclonic jerks time-locked to periodic spike/sharp wave discharges on hippocampal electroencephalography. The general opiate antagonist naloxone (NLX) (1 mg/kg, s.c.), another pharmacologic treatment with potential efficacy in dyskinesias or L-DOPA motor complications, produced similar seizures. No changes in anandamide levels in hippocampus and amygdala were found in convulsing NLX-treated BD rats. In contrast, NLX significantly increased anandamide levels in the same areas of normal uninfected animals, possibly protecting against seizures. Pretreatment with the anandamide transport blocker AM404 (20 mg/kg, i.p.) prevented NLX-induced seizures. These findings are consistent with an anticonvulsant role for endocannabinoids, counteracting aberrant firing produced by convulsive agents, and with a functional or reciprocal relation between opioid and cannabinoid tone with respect to limbic convulsive phenomena.

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.

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.

Local proliferation of microglia cells in response to neocortical injury in vitro.

Studies examined whether increased numbers of microglia following neural damage result from induced mitotic activity of resident microglia in situ. Organotypic slice cultures of neocortex were maintained for 1 week prior to placement of lesions. Increased numbers of OX-6 or tomato lectin labeled microglial cells were detected 1-8 days following lesions. Administration of BrdU to the cultures demonstrated lectin and BrdU double labeled microglial cells, conclusively demonstrating that a portion of the microglial cells were generated by local mitotic activity in situ.

AFGF promotes axonal growth in rat spinal cord organotypic slice co-cultures.

This study developed a slice culture model system to study axonal regeneration after spinal cord injury. This model was tested in studies of the roles of acidic fibroblast growth factor (aFGF) and peripheral nerve segments in axonal growth between pieces of spinal cord. Transverse sections of P15-P18 Sprague-Dawley rat spinal cord were collected for organotypic slice cultures. Group I consisted of two slices of spinal cord in contact with each other during the culture period. Group II consisted of two slices that were separated by 3 mm and connected by two segments of intercostal nerves. Group III consisted of single slices for studies of neuron survival. Some cultures from each group included aFGF in the culture medium. Bromodeoxyuridine (BrdU) was included in the medium for some cultures. The results showed three principal findings. First, counts of neurofilament-positive cells demonstrated that treatment with aFGF significantly increased the number of surviving neurons in culture. Second, neurofilament immunostaining and DiI tracing demonstrated axons crossing the junction between the two pieces of spinal cord or growing through the intercostal nerve segments, and these axons were seen only in cultures with aFGF treatment. Third, few cells were double stained for neurofilament and BrdU, and these were found only with aFGF treatment. These results demonstrate that (1) organotypic slice cultures present a useful model to study regeneration from spinal cord injury, (2) aFGF rescues neurons and promotes axonal growth in these cultures, and (3) segments of intercostal nerves promote axon growth between slices of spinal cord.