Induction of heterotopic and orthotopic cartilage and bone formation in mice

Heterotopic cartilage, bone and bone-marrow formation was achieved in mice by transplantation of a variety of xenogeneic established cell lines, by the transitional epithelium or by implants of demineralized bone matrix. The pattern and the sequence of events were always the same, regardless of the inducer used; viz., hyaline cartilage appeared 6-7 days after implantation, and endochondral bone formation followed. However, in cases of allogeneic implants of transitional epithelium into species other than the mouse, an intramembranous osteogenesis was the main mode of bone formation. When the yield of induced bone was high enough, a true myelopoiesis developed after three weeks. Heterotopically-induced bones had a relatively short life-span. Periosteal membranes of bones at the sites of sarcomes induced by M-MSV responded with rapid and extensive proliferation, with subsequent bone and, sometimes, hyaline cartilage deposition. This phenomenon was observed in long and cranial bones. However, bone induced heterotopically by demineralized bone matrix did not respond in such a way to the presence of M-MSV-induced sarcoma, suggesting that the connective tissue-encapsulated heterotopic bone was not a functioning periosteum. M-MSV-induced sarcoma also stimulates proliferation of elastic cartilage.     

Bone morphogenetic protein excipients: comparative observations on poloxamer

Clinicians await the availability of synthetic bioimplants that will replace the need for autogeneic bone grafts in bone reconstructive surgery. For more than a decade, researchers have evaluated delivery vehicles for the tissue morphogen bone morphogenetic protein. The object of this investigation was to measure induced bone development when bone morphogenetic protein was delivered by human tendon collagen, human demineralized bone matrix, hydroxyapatite, a composite of human tendon collagen and human demineralized bone matrix (tendon collagen + demineralized bone matrix), Poloxamer 407, and a composite of human demineralized bone matrix and Poloxamer 407. Sixty-three adult male Swiss Webster mice (Harlan Sprague-Dawley, Indianapolis, Ind.) received 126 implants. The animals were divided into seven groups of nine animals, depending on carrier (six carriers plus the positive control group) used. Each animal received a bone morphogenetic protein-enhanced carrier in one hindquarter muscle mass, with the contralateral leg being implanted with the carrier alone. Implants were evaluated by quantitative radiomorphometry validated by histologic methods. Radiographically, no significant differences were identified among any of the implants evaluated (p > 0.05). Histomorphometric analysis demonstrated that Poloxamer 407 was significantly (p < 0.05) better at delivering bone morphogenetic protein than the other carriers involved in this investigation. The new bone developed in a tubular or spherical shape. Interaction of endogenous and exogenous delivery systems seems to be essential for optimal transmission of bone morphogenetic protein. The importance of the excipient to deliver bone morphogenetic protein and develop a bone morphogenetic protein concentration gradient has been emphasized by other investigators and confirmed by our research on poloxamer. With further research on the physicochemical mechanisms of localization and transmission of bone morphogenetic protein, it may be possible to avoid hazardous operations with autogeneic bone.     

Accumulation, localization, and compartmentation of transforming growth factor beta during endochondral bone development

Endochondral bone formation was induced in postnatal rats by implantation of demineralized rat bone matrix. Corresponding control tissue was generated by implanting inactive extracted bone matrix, which did not induce bone formation. At various times, implants were removed and sequentially extracted with guanidine hydrochloride, and then EDTA and guanidine hydrochloride. Transforming growth factor beta (TGF beta) in the extracts was quantitated by a radioreceptor assay. TGF beta was present in demineralized bone matrix before implantation, and the concentration had decreased by 1 d after implantation. Thereafter, TGF beta was undetectable by radioreceptor assay until day 9. From day 9-21 the TGF beta was extracted only after EDTA demineralization, indicating tight association with the mineralized matrix. During this time, the content of TGF beta per milligram soluble protein rose steadily and remained high through day 21. This increased concentration correlated with the onset of vascularization and calcification of cartilage. TGF beta was detected only between days 3-9 in the controls; i.e., non-bone-forming implants. Immunolocalization of TGF beta in bone-forming implants revealed staining of inflammatory cells at early times, followed later by staining of chondrocytes in calcifying cartilage and staining of osteoblasts. The most intense staining of TGF beta was found in calcified cartilage and mineralized bone matrix, again indicating preferential compartmentalization of TGF beta in the mineral phase. In contrast to the delayed expression of TGF beta protein, northern blot analysis showed TGF beta mRNA in implants throughout the sequence of bone formation. The time-dependent accumulation of TGF beta when cartilage is being replaced by bone in this in vivo model of bone formation suggests that TGF beta may play a role in the regulation of ossification during endochondral bone development.     

Composition of cell-polymer cartilage implants

Cartilage implants for potential in vivo use for joint repair or reconstructive surgery can be created in vitro by growing chondrocytes on biodegradable polymer scaffolds. Implants 1 cm in diameter by 0.176 cm thick were made using isolated calf chondrocytes and polyglucolic acid (PGA). By 6 weeks, the total amount of glycosaminoglycan (GAG) and collagen (types I and II) increased to 46% of the implant dry weight; there was a corresponding decrease in the mass of PGA. Implant biochemical and histological compositions depended on initial cell density, scaffold thickness, and the methods of cell seeding and implant culture. Implants seeded at higher initial cell densities reached higher GAG contents (total and per cell), presumably due to cooperative cell-to-cell interactions. Thicker implants had lower GAG and collagen contents due to diffusional limitations.Implants that were seeded and cultured under mixed conditions grew to be thicker and more spatially uniform with respect to the distribution of cells, matrix, and remaining polymer than those seeded and/or cultured statically. Implants from mixed cultures had a 20-40-mum thick superficial zone of flat cells and collagen oriented parallel to the surface and a deep zone with perpendicular columns of cells surrounded by GAG Mixing during cell seeding and culture resulted in a more even cell distribution ad enhanced nutrient diffusion which could be related to a more favorable biomechanical environment for chondrogenesis. Cartilage with appropriate for and function for in vivo implantation ca thus be created by selectively stimulating the growth and differentiated function of chondrocytes (i.e., GAG and collagen synthesis) through optimization of the in vitro culture environment. (c) 1994 John Wiley & Sons, Inc.     

Effect of a freeze-dried CMC/PLGA microsphere matrix of rhBMP-2 on bone healing

The hypothesis of this research was that implants of poly(lactide-co-glycolide) (PLGA) microspheres loaded with bone morphogenetic protein-2 (rhBMP-2) and distributed in a freeze-dried carboxymethylcellulose (CMC) matrix would produce more new bone than would matrix implants of non-protein-loaded microspheres or matrix implants of only CMC. To test this hypothesis it was necessary to fashion microsphere-loaded CMC implants that were simple to insert, fit precisely into a defect, and would not elicit swelling. Microspheres were produced via a water-in-oil-in-water double-emulsion system and were loaded with rhBMP-2 by soaking them in a buffered solution of the protein at a concentration of 5.4 mg protein per gram of PLGA. Following recovery of the loaded microspheres by lyophilization matrices for implantation were prepared by lyophilizing a suspension of the microspheres in 2% CMC in flat-bottom tissue culture plates. Similar matrices were made with 2% CMC and with 2% CMC containing blank microspheres. A full-thickness calvarial defect model in New Zealand white rabbits was used to assess bone growth. Implants fit the defect well allowing for direct application. Six weeks postsurgery, defects were collected and processed for undecalcified histology. In vitro, 60% of the loaded rhBMP-2 released from devices or microspheres in 5 to 7 days. With the unembedded microspheres releasing faster than those embedded in CMC In vivo. the rhBMP-2 microspheres greatly enhanced bone healing, whereas nonloaded PLGA microspheres in the CMC implants had little effect. The results showed that a lyophilized device of rhBMP-2 PLGA microspheres in CMC was an effective implantable protein-delivery system for the use in bone repair. Published: October 7. 2001.     

Immunohistochemical detection of an enamel protein-related epitope in rat bone at an early stage of osteogenesis

Monoclonal antibody MI315 was produced against hamster tooth germ homogenate by in vitro immunization. It was found that MI315 reacted with enamel matrix, ameloblasts, and bone matrix at an early stage of osteogenesis. Decalcified tissues of rat femurs and mandibles were examined with MI315 using indirect immunofluorescence. In endochondral ossification of femurs, immunoreactivity was found in bone extracellular matrix (ECM) deposited on the surface of the cartilage core of primary spongiosa, but not in the cartilage core itself. In intramembranous ossification of 0-day-old rat mandibles, intense immunofluorescence was detected in bone ECM and a few young osteocytes, but not in osteoblasts. Immunoreactivity in bone ECM of 2-day-old rats decreased and almost disappeared from bone ECM of 4-day-old rats. Although in nondecalcified sections of 0-day-old rats, negligible immunofluorescence was detected in bone ECM which showed positive staining in decalcified tissues, the immunostaining appeared after decalcification using ethylenediaminetetraacetic acid (EDTA). These results indicate that a substance(s), which had a common epitope with an enamel-derived protein(s), existed in immature bone ECM of both endochondral and intramembranous ossification, and that it might be masked by bone mineral. Monoclonal antibody MI315 is a useful tool to investigate the time- and position-specific changes in osteogenesis and amelogenesis.     

The induction of bone by an osteogenic protein and the conduction of bone by porous hydroxyapatite: a laboratory study in the rabbit.

The influence of the addition of an osteoinductive protein, capable of inducing extraskeletal ossification, on bone ingrowth into coralline porous hydroxyapatite was evaluated in the rabbit using a calvarium onlay model. Twenty-three rabbits received hydroxyapatite implants (10 x 10 x 2 mm) prepared with and without osteoinductive protein. These were implanted on the frontal bone and secured by wire fixation after 0.25 mm of the cortical surface was abraded. The implants were harvested at 3 and 4 months and analyzed for percentage of bone ingrowth by histologic examination of decalcified H&E sections and by scanning electron microscope backscatter image analysis. The osteoinductive protein-treated implants demonstrated significantly greater amounts of bone ingrowth at both 3 (52.0 versus 10.3 percent; p less than 0.001) and 4 months (66.1 versus 39.2 percent; p less than 0.005) than the untreated implants. The type of bone found in all osteoinductive protein-treated implants was predominantly lamellar. Untreated implants contained mostly woven bone at 3 months, with increasing amounts of lamellar bone appearing at 4 months. These results suggest that the combination of a bone-inducing protein and a suitable osteoconductive matrix may provide an alternative to bone grafting.     

Recombinant mink cell focus-inducing virus and long terminal repeat alterations accompany the increased leukemogenicity of the Mo+PyF101 variant of Moloney murine leukemia virus after intraperitoneal inoculation.

We recently showed that different routes of inoculation affect the leukemogenicity of the Mo+PyF101 variant of Moloney murine leukemia virus (M-MuLV). Intraperitoneal (i.p.) inoculation of neonatal mice with Mo+PyF101 M-MuLV greatly enhanced its leukemogenicity compared with subcutaneous (s.c.) inoculation. We previously also suggested that the leukemogenicity defect of Mo+PyF101 M-MuLV when inoculated s.c. may result from the inability of this virus to form env gene recombinant (mink cell focus-inducing [MCF]) virus. In this study, virus present in end-stage tumors and in preleukemic animals inoculated i.p. by Mo+PyF101 M-MuLV was characterized. In contrast to s.c. inoculation, all tumors from i.p.-inoculated mice contained high levels of recombinant MCF virus. Furthermore, Southern blot analyses demonstrated that the majority of the tumors contained altered Mo+PyF101 M-MuLV long terminal repeats. The U3 regions from several tumors with altered long terminal repeats were cloned by PCR amplification. Sequence analyses indicated that the M-MuLV 75-bp tandem repeat in the enhancer region was triplicated. This amplification was also previously observed in mice infected s.c. with a pseudotypic mixture of Mo+PyF101 M-MuLV and Mo+PyF101 MCF virus. The enhancer triplication was an early event, and it occurred within 2 weeks postinfection. Recombinant MCF viruses were not detected by Southern blot analyses until 4 weeks postinfection. Thus, the M-MuLV enhancer triplication event was initially important for efficient propagation of ecotropic Mo+PyF101 M-MuLV. The increased leukemogenicity following i.p. inoculation could be explained if the triplication enhances Mo+PyF101 M-MuLV replication in the bone marrow and bone marrow infection is required for recombinant MCF virus formation.     

Circadian-phase-dependency in [3H]-dihydroalprenolol binding to rat heart ventricular membranes

Binding studies with the beta-adrenoceptor antagonist ligand [3H]-dihydroalprenolol ([3H]-DHA, spec. act, 90-102 Ci/mmol) were performed with ventricular membranes L-D-synchronized (L:0.7-19 hr, D:-07 hr) male rats, sacrificed either at 08 hr or at 20 hr. Saturation experiments with crude or washed and preincubated membranes revealed two affinity states of specific [3H]-DHA binding which were abolished after addition of the guanine nucleotide Gpp(NH)p. In crude membranes the apparent Bmax-value at 20 hr was about 40% higher than at 08 hr, in washed and preincubated membranes the nocturnal increase in the apparent Bmax-value was not observed. Pretreatment of rats with isoprenaline (50 mg/kg, i.p.) decreased and catecholamine depletion (reserpine plus inhibition of tyrosine-hydroxylase) increased Bmax-values in crude membranes. The circadian-stage-dependent and the drug-induced effects on the apparent number of beta-adrenoceptors are assumed to be due to circadian or drug-induced variations in the turnover of cardiac noradrenaline.     

Hematopoiesis on cellulose ester membranes (CEM). XII. Effect of membrane enrichment by purified matrix proteins

Cellulose ester membranes (CEM) were enriched with the following purified matrix proteins: collagen I, II, IV, proteoglycan and laminin. Fifteen milligrams of each were placed on CEM which were then folded into open-ended tubes implanted i.p. and s.c. CEM were removed after 3, 6 and 12 months and examined histologically. There was no evidence of hematopoiesis or new bone formation on the implanted, enriched CEM at any of the intervals examined. Collagen I and proteoglycan-enriched CEM showed evidence of increased sinusoid-like vascular structures.     

Pentadecapeptide BPC 157 attenuates gastric lesions induced by alloxan in rats and mice

A diabetogenic alloxan regimen produced lesions in all stomachs of treated animals, either rats (200 mg x kg(-1) s.c.) or mice (400 mg x kg(-1) i.p.). In control animals, the lesions, when developed (i.e. 24 h following application), appear to be quite sustained, and consistently present also after 1 or 2 weeks. The application of the pentadecapeptide BPC 157 (10 microg or 10 ng x kg(-1) i.p. coadministered together with alloxan) would significantly attenuate these lesions' appearance. This beneficial effect seems to be present in either rats or mice and in either of the tested intervals. Importantly, the beneficial effect seems to be shared by both microgram and nanogram regimens.     

Antitumor effects of human recombinant macrophage colony-stimulating factor against rat brain tumors

The tumoricidal effects of M-CSF were examined using two subcutaneously-transplanted rat brain tumor cell lines, 9L and T9 gliomas. In rats treated with high-dose M-CSF (16 million U/kg administered for 4 days a week for 3 weeks), 9L glioma growth was inhibited by 81.9% following subcutaneous (s.c.) injection and by 70.5% after intraperitoneal (i.p.) injection and T9 glioma growth was inhibited by 69.2% after i.p. injection. After short-term treatment with high-dose M-CSF (32 million U/kg administered s.c. for 6 consecutive days, 9L glioma growth was inhibited by 82.1%. All these inhibitory effects differed significantly compared with the respective untreated control groups. However, treatment with low-dose M-CSF (1.6 million U/kg administered s.c. for 4 days a week for 3 weeks) showed no significant effects against 9L and T9 glioma growth compared with the untreated controls. No significant effects of M-CSF against cell proliferation, measured as PCNA expression, were observed in any group. Significant hematopoietic effects on the leukocyte counts were observed only in the groups treated with high dose M-CSF. These results suggest that M-CSF at a high dose which produces hematopoietic effects on peripheral leukocytes inhibits the growth of gliomas. This inhibitory effect may have been due to a tumoricidal mechanism of M-CSF that depended on the production or release of some hematopoietic soluble factors, but was independent of PCNA expression by the tumors.Abbreviations BBB blood-brain barrier - G-CSF granulocyte colony-stimulating factor - GM-CSF granulocyte-macrophage colony-stimulating factor - hM-CSF human macrophage colony-stimulating factor - IFN interferon - IL-1 interleukin-1 - IL-6 interleukin-6 - M-CSF macrophage colony-stimulating factor - PCNA proliferating cell nuclear antigen - rhM-CSF recombinant human macrophage colony-stimulating factor - TNF tumor necrosis factor     

The leukemogenic potential of an enhancer variant of Moloney murine leukemia virus varies with the route of inoculation.

We previously showed that the Mo+PyF101 variant of Moloney murine leukemia virus (M-MuLV) is poorly leukemogenic when inoculated subcutaneously (s.c.) into neonatal mice. We recently found that intraperitoneal (i.p.) inoculation of neonatal mice with the same virus significantly enhanced its leukemogenicity. In this study, infections of neonatal mice by the two different routes of inoculation were compared. We studied replication of the virus in vivo to identify critical preleukemic events. These would be observed in mice inoculated i.p. by Mo+PyF101 M-MuLV but not when inoculation was s.c. Infectious center assays indicated that regardless of the route of inoculation, Mo+PyF101 M-MuLV showed delayed infection of the thymus compared with wild-type M-MuLV. On the other hand, i.p.-inoculated mice showed more rapid appearance of infectious centers in the bone marrow than did s.c.-inoculated animals. Thus, the enhanced leukemogenicity of i.p. inoculation correlated with efficient early infection of the bone marrow and not with early infection of the thymus. These results suggest a role for bone marrow infection for efficient leukemogenesis in Mo+PyF101 M-MuLV-infected mice. Consistent with this notion, if bone marrow infection was decreased by injecting 10- to 12-day-old animals i.p., leukemogenicity resembled that of s.c. inoculation. Thus, two cell types that are critical for the induction of efficient leukemia were implicated. One cell delivers virus from the site of s.c. inoculation (the skin) to the bone marrow and is apparently restricted for Mo+PyF101 M-MuLV replication. The second cell is in the bone marrow, and its early infection is required for efficient leukemogenesis.     

DNA vaccination against viral haemorrhagic septicaemia (VHS) in rainbow trout: size,dose, route of injection and duration of protection-early protection correlates with Mx expression

Rainbow trout of different sizes (10 and 100g) were injected intramuscularly (i.m.) or intraperitoneally (i.p.) with different doses (range 10 ng-10 microg) of a viral haemorrhagic septicaemia (VHS)-DNA vaccine (pcDNA3vhsG). As controls, fish were injected with the pcDNA3 plasmid alone, or with inactivated VHS virus. Fish were challenged at different times post-vaccination (p.v.) to assess protection. At certain times p.v., serum samples were analysed for neutralising antibody and liver tissue was analysed for Mx mRNA expression. A DNA dose of 0.5 microg injected by the i.m. route induced protection in fish of all sizes in challenges performed either 1 or 4 weeks p.v. This dose also conferred effective protection up to 9 months p.v. in fish >100 g. With lower doses of DNA (0.1 and 0.01 microg) and challenge at 4 weeks p.v., 10 g fish were partially protected but protection was not observed in 100 g fish. Vaccination by the i.p. route induced no or lower levels of protection compared with the i.m. route. Fish vaccinated with 0.5 microg DNA i.m. had no detectable serum neutralising antibody (NAb) at 4 weeks p.v. (with the exception of a single 10 g fish) but antibody was detected at 8 weeks and 6 months p.v. but not at 9 months p.v. However, cohorts of these fish showed effective protection at all timepoints. Lack of detectable levels of NAb (at 9 weeks p.v.) despite partial protection in challenge at 4 weeks p.v. was also observed with 0.01 microg doses of DNA i.m. NAb was detected in sera of fish at 8 weeks after vaccination with 0.1 microg i.m. but not in fish vaccinated with doses of 0.01-0.5 microg i.p. Early protection (1 week p.v.) correlated with elevated Mx gene expression.     

Effectiveness of fresh frozen and cryopreserved homologue iliac crest grafts used in sinus lifting: a comparative study

In the last decade, several investigators have reported that autologous and homologous fresh frozen bones (FFB) are effective materials to restore alveolar ridges previous to insert dental implants. Recently we have used cryopreserved homologue grafts (CFFB). Here we reported a retrospective comparative study between implants inserted in FFB and CFFB evaluate their clinical outcome. Patients were treated with a split mouth scheme for bone grafting with FFB and CFFB and spiral family implants (SPI) were inserted in the same surgical time. Several variables (patient, grafts, anatomic site, implant, prosthetic restoration) were investigated. Implant’ failure and peri-implant bone resorption were considered as predictor of clinical outcome. A total of 84 SFIs were inserted in 12 patients. Implants were inserted to replace 8 incisors, 4 cuspids, 31 premolars and 41 molars. The mean follow-up was 14 months. Three out of 84 implants was lost (i.e. survival rate SVR = 96.4%) and no differences were detected among the studied variables. Similar result was obtained by analyzing the crestal bone resorption around implant’ neck (i.e. success rate). FFB and CFFB have high and comparable survival and success rate. Implants inserted with one step surgical procedure in native (i.e. not grafted) bone, FFB and CFFB have similar clinical outcome.     

Appearance of Mink Cell Focus-Inducing Recombinants during In Vivo Infection by Moloney Murine Leukemia Virus (M-MuLV) or the Mo+PyF101 M-MuLV Enhancer Variant: Implications for Sites of Generation and Roles in Leukemogenesis

One hallmark of murine leukemia virus (MuLV) leukemogenesis in mice is the appearance of env gene recombinants known as mink cell focus-inducing (MCF) viruses. The site(s) of MCF recombinant generation in the animal during Moloney MuLV (M-MuLV) infection is unknown, and the exact roles of MCF viruses in disease induction remain unclear. Previous comparative studies between M-MuLV and an enhancer variant, Mo+PyF101 MuLV, suggested that MCF generation or early propagation might take place in the bone marrow under conditions of efficient leukemogenesis. Moreover, M-MuLV induces disease efficiently following both intraperitoneal (i.p.) and subcutaneous (s.c.) inoculation but leukemogenicity by Mo+PyF101 M-MuLV is efficient following i.p. inoculation but attenuated upon s. c. inoculation. Time course studies of MCF recombinant appearance in the bone marrow, spleen, and thymus of wild-type and Mo+PyF101 M-MuLV i.p.- and s.c.-inoculated mice were carried out by performing focal immunofluorescence assays. Both the route of inoculation and the presence of the PyF101 enhancer sequences affected the patterns of MCF generation or early propagation. The bone marrow was a likely site of MCF recombinant generation and/or early propagation following i.p. inoculation of M-MuLV. On the other hand, when the same virus was inoculated s.c., the primary site of MCF generation appeared to be the thymus. Also, when Mo+PyF101 M-MuLV was inoculated i.p., MCF generation appeared to occur primarily in the thymus. The time course studies indicated that MCF recombinants are not involved in preleukemic changes such as splenic hyperplasia. On the other hand, MCFs were detected in tumors from Mo+PyF101 M-MuLV s. c.-inoculated mice even though they were largely undetectable at preleukemic times. These results support a role for MCF recombinants late in disease induction.     

Placement of endosteal implants in the zygoma after maxillectomy: a Cadaver study using surgical navigation

Endosteal implants facilitate obturator prosthesis fixation in tumor patients after maxillectomy. Previous clinical studies have shown, however, that the survival of implants placed into available bone after maxillectomy is generally poor. Nevertheless, implants positioned optimally in residual zygomatic bone provide superior stability from a biomechanical point of view. In a pilot study, the authors assessed the precision of VISIT, a computer-aided surgical navigation system dedicated to the placement of endosteal implants in the maxillofacial area. Five cadaver specimens underwent hemimaxillectomy. The cadaver head was matched to a preoperative high-resolution computed tomograph by using implanted surgical microscrews as fiducial markers. The position of a surgical drill relative to the cadaver head was determined with an optical tracking system. Implants were placed into the zygomatic arch, where maximum bone volume was available. The results were assessed using tests for localization accuracy and postoperative computed tomographic scans of the cadaver specimens. The localization accuracy of landmarks on the bony skull was 0.6 +/- 0.3 mm (average +/- SD), as determined with a 5-df pointer probe; the localization accuracy of the tip of the implant burr was 1.7 +/- 0.4 mm. The accuracy of the implant position compared with the planned position was 1.3 +/- 0.8 mm for the external perforation of the zygoma and 1.7 +/-1.3 mm for the internal perforation. Eight of 10 implants were inserted with maximal contact to surrounding bone, and two implants were located unfavorably. Reliable placement of implants in this region is difficult to achieve. The technique described in this article may be very helpful in the management of patients after maxillary resection with poor support for obturator prostheses.     

Forebrain implants of estradiol stimulate maternal nest-building in ovariectomized rabbits

In rabbits, estradiol and progesterone (P) stimulate digging a maternal burrow while P withdrawal promotes straw-carrying. To investigate where such hormones act to regulate those activities, ovariectomized rabbits were implanted with estradiol benzoate (EB; Experiment 1) in the nucleus accumbens (ACC), the principal nucleus of the medial preoptic area or the dorsal hippocampus. Implants were combined with s.c. P injections. In Experiment 2, P (in crystals or dissolved in oil) was implanted in the same regions as in Experiment 1, combined with s.c. injections of EB. Implants of EB into the ACC or MPOA-bed nucleus of the stria terminalis (BNST) stimulated significant digging across the period of P injections in 72% and 67% of females, respectively. Neither EB implants in the hippocampus nor cholesterol implants in the MPOA-BNST were effective in eliciting digging. P withdrawal provoked a rapid decline of digging in all animals; it also stimulated straw-carrying in 53% of females implanted with EB in the MPOA-BNST. P implants failed to stimulate digging in most females injected with EB. Removal of P crystals did not promote straw-carrying. Results support an action of estradiol on the ACC and MPOA-BNST to promote digging while only the MPOA-BNST is involved in stimulating straw-carrying. The failure of P implants to stimulate digging or straw-carrying in EB-treated females suggests that the stimulation of other or additional brain areas by P is necessary to fully activate maternal nest-building.     

Developmental expression of the putative transcription factor Egr-1 suggests that Egr-1 and c-fos are coregulated in some tissues

We have investigated developmental expression of the gene Egr-1, which encodes a protein containing three zinc fingers. Egr-1 like c-fos is a serum inducible, early response gene, which is co-induced with c-fos in a variety of quite different situations. A single 3.7-kb RNA was detected throughout fetal mouse development, which increased in absolute levels in total fetal RNA from 9.5 to 12.5 days post coitum (p.c.). In situ hybridization to 14.5- and 17.5-day p.c. fetal tissues demonstrated Egr-1 accumulation at several specific sites. These included mesenchymal components of the developing tooth germs and salivary and nasal glands; an ectodermally derived component of the whisker pad and developing muscle, cartilage, and bone. Expression of Egr-1 in cartilage and bone showed a strikingly similar expression to previously published reports of c-fos in these tissues. High levels of Egr-1 RNA was observed at the perichondrial interface of opposing cartilaginous elements and in interstitial cells that lie in between. Bone expression was observed in membranous bone of the head, alveolar bone around the tooth germs, and at periosteal and endochondral ossification sites in the limb bones. Our data support the idea that Egr-1 and c-fos may be coregulated in vivo and together may regulate normal development of the skeleton.     

Complex role of 5-HT in the regulation of TSH secretion in the male rat

The role of 5-hydroxytryptamine (5-HT) in the regulations of TSH secretion was studied in male rats using both peripheral and central administration of the drugs. Basal TSH levels were not modified by moderate doses of 5-HT (subcutaneously) or its precursors or antagonists (intraperitoneally) given 1 h before decapitation. The cold-stimulated TSH secretion was decreased by L-tryptophan (L-TRP, 400 mg/kg i.p.), quipazine (10 mg/kg i.p.) and 5-HT (1 or 5 mg/kg s.c. or i.v.) as well as by p-chlorophenylalanine (pCPA, 20 or more mg/kg i.p.) when the drugs were given 1 h before sampling. pCPA (100-400 mg/kg i.p.) was active 24-48 h after the injection but repetitive administration did not affect TSH levels. 5-HT (5 mg/kg s.c.) was effective also in pinealectomized animals. L-TRP and 5-hydroxytryptophan potentiated the TRH-stimulated TSH secretion when given 1 h before killing. 5-HT (10 microgram/rat) infused into the third ventricle enhanced the cold-stimulated TSH secretion when given 30-45 min before sampling. When injected into the medial basal hypothalamus, 50-HT (1-10 microgram/rat) had no effect on basal or stimulated TSH levels. The results suggest: (1) 5-HT does not play any role in the regulation of basal TSH secretion; (2) in the cold-stimulated TSH secretion 5-HT has a stimulatory action evidently inside the blood-brain barrier and also an inhibitory effect obviously outside this barrier.