Intra-operative diabetes insipidus associated with cervical spine traction during staged scoliosis surgery.

A 15-year-old boy with cerebral palsy and epilepsy presented for a posterior spinal fusion as part of staged repair of thoracolumbar scoliosis. Total intravenous anaesthesia was induced and maintained with propofol, remifentanil and ketamine. Following prone positioning, cervical traction was applied. Polyuria developed intra-operatively, from 4 to 18 ml.kg-1.h-1. There was a corresponding rise in plasma sodium concentration from 132 to 145 mmol.l-1. Haemodynamic stability was maintained with boluses of Hartmann's solution and a noradrenaline infusion. Given the possibility of diabetes insipidus due to reduced cerebral perfusion pressure, the cervical traction was removed. This initially showed a good response with a transient reduction in polyuria to 3 ml.kg-1.h-1 before rising to 8 ml.kg-1.h-1. Subsequently, a vasopressin infusion was started with normalisation of diuresis and plasma sodium concentration by the end of surgery. Diabetes insipidus is an endocrine disorder related to lack of production or insensitivity to vasopressin. In the peri-operative period, it is mainly associated with pituitary surgery and rarely with spinal surgery. To the authors' knowledge, this is only the second report of diabetes insipidus associated with staged scoliosis surgery. Cervical traction should be considered as a potential cause of intra-operative diabetes insipidus.

Cerebrospinal fluid levels of glial cell-derived neurotrophic factor correlate with spinal cord stimulation frequency in patients with neuropathic pain: a preliminary report.

STUDY DESIGN: Case series. OBJECTIVES: To evaluate relationships between spinal cord stimulation (SCS) parameters and levels of glial cell-derived neurotrophic factor (GDNF). SETTING: Ambulatory pain clinic of St James's Hospital, Dublin, Ireland. METHODS: Nine patients with an implanted SCS and Failed Back Surgery Syndrome (FBSS) were administered the Brief Pain Inventory and Short Form (36) Health Survey. Following a lumbar puncture, levels of GDNF in cerebrospinal fluid (CSF) were assayed and correlated with stimulation parameters. Controls were patients with arthritic back pain who were matched for age, gender and SF-36 score. RESULTS: Concentrations of GDNF in CSF are higher in patients with FBSS than controls (P=0.002) and correlate with SCS frequency (P=0.029). CONCLUSION: Concentrations of GDNF in CSF are higher in neuropathic pain and appear to be related to stimulation frequency. Further work is needed to evaluate this potential relationship, both in neuropathic pain and in other contexts such as locomotor dysfunction.

Population size and radiosensitivity of murine hematopoietic endogenous long-term repopulating cells.

A new assay is described that measures the numbers (n) of endogenous long-term repopulating cells (eLTRC) surviving sublethal irradiation. The assay is based on analysis of variances of Pgk-1 phenotypes within groups of sublethally irradiated Pgk-1(a/b) mice. When ln n is plotted as a function of dose, the radiosensitivity (D0) is the negative reciprocal of the slope and the gamma-intercept is N or total numbers of eLTRC per mouse. The assay is unique in that N is an absolute value not requiring correction for seeding efficiency, or f. From two independent estimates, total numbers of eLTRC were determined to be 8,700 or 11,900 cells per mouse and D0 was found to be 0.82 or 0.83 Gy. If eLTRC are, in fact, the long-term repopulating cells (LTRC) defined by classical transfer assay, then LTRC can home to the marrow with nearly unit efficiency, only one to two LTRC are required for a mouse to survive a radiation LD50/30, and LTRC possess nearly unlimited self-renewal potential.

Cytokine gene expression after in vivo primary immunization with goat antibody to mouse IgD antibody.

Cytokines are important mediators of effector lymphoid cell function during an immune response, but their expression during an in vivo immune response has not been well documented. We analyzed the kinetics of cytokine gene expression during the course of an in vivo primary immune response to goat antibody to mouse IgD antibody. Total RNA was purified from spleens taken from freshly killed BALB/c mice 1 to 7 days after immunization. The reverse transcriptase polymerase chain reaction was used to evaluate the expression of seven cytokine genes, all of which encode cytokines that are secreted by T cells and are important in T and/or B cell activation and differentiation. These were IFN-gamma, IL-2, IL-4, IL-5, IL-6, IL-9, and IL-10. IL-2 and IL-9 exhibited an early elevated expression at days 2 to 3, and declined as the expression of IL-4, IL-6, IL-10, and IFN-gamma increased. In contrast, IL-5 gene expression showed little change, exhibiting a similar pattern to the housekeeping gene, hypoxanthine-guanine phosphoribosyl transferase. Cell sorting of CD4+ and CD4- cells at day 3 and day 5 after immunization revealed that CD4+ cells were the predominant source of the elevated cytokines (with the exception of IL-6). Our results demonstrate a specific and highly reproducible cytokine gene expression pattern during the course of a primary in vivo immune response that is marked by an absence of a clear-cut Th1/Th2 dichotomy.

Interaction of lymphocytes and high endothelial venules in irradiated lymph nodes.

After total-body exposure to various doses of ionizing radiation, the ability of lymphocytes to interact specifically with high endothelial venules of rat cervical and mesenteric lymph nodes was analyzed in frozen sections. Following a radiation dose of 1.5 Gy, high endothelial venules remained intact and the binding of unirradiated lymphocytes to the venules was enhanced relative to unirradiated controls. At radiation doses above 5.0 Gy, damage to high endothelial venules was observed histologically as well as assessed functionally. There was a significant decrease in specific lymphocyte-venule binding and a significant increase in nonspecific binding. These findings suggest that radiation-induced damage to high endothelial venules might play a role in radiation-induced immunosuppression by interfering with the normal passage of lymphocytes from the blood into lymph nodes via a specific interaction between lymphocytes and high endothelial venules.

Further enrichment and analysis of rat CFU-s.

Using the monoclonal antibody W3/13, which recognizes a determinant expressed on a sialoglycoprotein, rat marrow cells with the phenotype Thy-1 antigen upper 20% positive (Ox720) and high molecular weight leukocyte common antigen negative (Ox22-) were separated into W3/13 dim (W3/13d) and W3/13 bright (W3/13b) subpopulations by single-laser cell sorting. The spleen colony-forming unit (CFU-s) was found in the W3/13d fraction. A 468-fold enrichment of CFU-s was achieved. Only 20% of the Ox720, Ox22-, and W3/13d cells were in the S phase of the cell cycle as compared to 56% of Ox720, Ox22-, and W3/13b cells. Using Indo-1, it was not possible to demonstrate increases in cytosolic Ca++ levels within the enriched CFU-s population by colony-stimulating factors (CSFs) or interleukins 1, 2, and 3. However, challenge with the Ca++ ionophore, ionomycin, demonstrated apparent heterogeneity of intracellular Ca++ management within the enriched CFU-s population. The source of this heterogeneity is not known. Only a 12-day CFU-s was detected in the rat, and it was predominantly, but not exclusively, a Rhodamine 123 (Rh123) dull cell.

Flow cytometry techniques in radiation biology.

Hematopoietic stem cells (HSC) are present in the marrow at a concentration of approximately 2-3 HSC per 1000 nucleated marrow cells. In the past, only clonogenic assays requiring 8-13 days and ten irradiated recipient rodents were available for assaying HSC. Because of the importance of HSC in the postirradiation syndrome, we have developed a new rapid method based on flow cytometry not only to assay but also to purify and characterize HSC. This new method makes extensive use of monoclonal antibodies conjugated to fluorescent phycobiliproteins through the sulfhydryls of the hinge region of the IgG molecule. An optical bench arrangement with a dye laser and an argon laser was used for dual excitation of the phycobiliprotein-monoclonal antibody conjugates and various cellular and DNA probes. Using 4',6-diamidino-2-phenylindole dihydrochloride (DAPI) exclusion to identify viable cells, it was possible to follow regeneration of postirradiated rat marrow HSC.

Characterization of rat prothymocyte with monoclonal antibodies recognizing rat lymphocyte membrane antigenic determinants.

Utilizing the technique of fluorescence-activated cell sorting and monoclonal antibodies directed at rat membrane antigens, various subpopulations of Lewis bone marrow cells were isolated and subsequently transfused into sublethally irradiated, histocompatible NBr recipient rats by either intravenous or intrathymic inoculation. Recipient rats were sacrificed and cell suspensions from thymus and other lymphoid tissue were examined for the presence of the RT7.1 marker on Lewis thymus-derived lymphocytes by fluorescence-activated cell analysis. From these studies, the population of Lewis bone marrow cells that could reconstitute T cells in the NBr rats was found to be Ox-22 negative, Ox-7 positive, W3/13 positive, and Ox-18 positive. Further analysis characterized the prothymocyte as being Ox-7 upper 20% positive and W3/13 weakly positive. In addition this marrow cell population was able to protect lethally irradiated Lewis rats (9.5 Gy) in 30-day survival tests. These studies have indicated that the prothymocyte either has been derived from the Ox-22 negative, Ox-7 upper 20% positive, and W3/13 positive marrow cells or, like the hematopoietic stem cell, this cell has also been characterized by this phenotype.

New bone formation and bone marrow differentiation induced in rats by extracellular bone matrix implantation: effect of local preirradiation on the process.

Subcutaneous implantation of demineralized diaphyseal rat bone matrix in ACI rats initiates a developmental cascade that results in the formation of new endochondral bone and an associated hematopoietic bone marrow differentiation. Irradiation (1500 rad, 60Co) of the implantation site 24 h prior to implantation suppresses the formation of endochondral bone and bone marrow. All phases of the developmental cascade, including chemotaxis, proliferation, and differentiation, are arrested by the irradiation. Simultaneous implantation with the extracellular matrix of bone marrow, bone, pieces of a four-day-old implant or of thoracic muscle--but not of brain, liver, or spleen tissue--results in the development of endochondral bone and bone marrow at the irradiated site. Concurrent implantation with the extracellular matrix of in vitro growing fibroblasts of marrow or ossicle origin does not restore the developmental cascade.

Purification and analysis of rat hematopoietic stem cells by flow cytometry.

The monoclonal antibody OX7 recognizes an epitope expressed on the Thy-1 glycoprotein, OX22 recognizes the high molecular weight form(s) on leukocyte common antigen, and W3/13 recognized determinants found on certain sialoglycoproteins. Recently, the rat colony-forming unit spleen (CFU-S) was characterized as being OX7 upper 20% positive (OX7u20%), OX22 negative (OX22-), and W3/13 weakly positive (W3/13+). In the present study these observations have been extended to include the hematopoietic stem cell (HSC). Rat marrow cells were incubated with allophycocyanine-OX7 Fab' (APC-OX7 Fab') and phycoerythrin B-OX22 Fab' (Phy B-OX22 Fab'). The cells were sorted with a FACS-II instrument by using a Krypton laser tuned to the 530 nm spectral line for phycobiliprotein excitation. It was found that marrow cells capable of protecting lethally irradiated Lewis rats (9.5 Gy total body radiation, 0.4 Gy/min Co60) had the phenotype OXu20%, OX22-. The percentage of cells in the marrow with this phenotype was found to be 0.34 +/- 0.01 (mean +/- S.E.). Three thousand of these cells were required to rescue 50% of lethally irradiated recipients (30-d survival), while the number of unsorted bone marrow cells required was 1.05 X 10(6). Thus, a 350-fold purification of the HSC was realized. Although CFU-S copurified with HSC, purification of only 105-fold was obtained. This might indicate that purified HSC have a reduced capacity to generate splenic hematopoietic colonies. The OX7u20%, OX22- -enriched HSC population could be further divided into W3/13 dim and W3/13+ subpopulations by three-parameter immunofluorescence analysis with the use of a new optical bench arrangement.

Rat colony-forming unit spleen is OX7 positive, W3/13 positive, OX1 positive, and OX22 negative.

Using the monoclonal antibodies OX7 HL, W3/13 HLK, OX1 HLK, OX22, and the technique of fluorescence activated cell sorting, it was possible to characterize the phenotype of the rat marrow CFU-S as OX7 upper 20% positive, W3/13 lower 50% positive, OX1 positive, and OX22 negative. OX7 recognizes an antigenic determinant expressed on the Thy1 glycoprotein, W3/13 recognizes a determinant expressed on some sialoglycoproteins, OX1 recognizes all four apparent molecular weight forms of leukocyte common antigen, while OX22 recognizes only the high molecular weight forms of leukocyte common antigen. It was determined that the concentration of OX7 upper 20% positive, W3/13 lower 50% positive, OX1 positive, and OX22 negative cells in the marrow was 3085 +/- 1446/10(6) cells. For comparison, the calculated concentration of marrow stem cells using a 2-h seeding efficiency was found to be 501 CFU-S/10(6) cells and, using a 24-h seeding efficiency, it was found to be 1415 CFU-S/10(6) cells. Although requiring further refinements, these results suggest that an assessment of CFU-S marrow concentration might be achieved using multiparameter flow cytometry. Also, a technique for the conjugation of the Fab' fragment of the monoclonal antibody OX7 to phycoerythrin is described.

Effect of sublethal ionizing radiation on rat Peyer's patch lymphocytes.

After sublethal doses of ionizing radiation, rat Peyer's patch lymphocytes regenerated significantly more slowly than lymphocytes from spleen, thymus, and peripheral lymph nodes. Long Evans rats were exposed to 150 rad (40 rad/min) of whole-body irradiation from a 60Co, gamma-emitting source. On Days 1-20 postirradiation, single cell suspensions of lymphocytes from thymus, spleen, peripheral lymph nodes, and Peyer's patches were stained with mouse monoclonal antibody reagents specific for rat lymphocyte subpopulations (Ia+ cells, non-helper T-cell subsets, and helper T-cell subsets). Cells were then counterstained with Texas Red-conjugated, goat anti-mouse IgG and, at the same time, were also stained with fluorescein diacetate to determine viable lymphocytes. The stained lymphocytes were analyzed using a dual-laser, fluorescent-activated cell sorter (Becton-Dickinson FACS-II) from which the percentage of each lymphocyte subpopulation was determined. From our studies, we found that all subpopulations of lymphocytes were affected similarly by irradiation. In addition, we observed that viable lymphocyte subpopulation in thymus, spleen, and peripheral lymph nodes from irradiated animals returned to normal (nonirradiated control animals) levels 5-12 days postirradiation, while viable lymphocyte subpopulations in Peyer's patches from irradiated animals remained suppressed up to 20 days postirradiation. These results suggest that either the lymphocytes or, more likely, the microenvironment of Peyer's patches is more greatly damaged by ionizing radiation than that observed in other lymphoid tissue.

Establishment of the hematopoietic microenvironment in the marrow of matrix-induced endochondral bone.

Implantation of demineralized diaphyseal bone matrix (DBM) into subcutaneous sites in allogeneic Long-Evans rats results in new endochondral bone formation accompanied by hematopoietic bone marrow differentiation in the newly formed ossicles. In the present study, we investigated the relationship between the time of appearance of hematopoietic stem cells (CFU-S) and those of certain postulated elements of the hematopoietic microenvironment, i.e., fibroblast colony-forming cells (CFC-F), colony-stimulating factors (CSF), and collagen types. CFU-S were first detected at 16 days' postimplantation in the developing ossicle. Their numbers increased exponentially until day 24, decreased slightly between days 27 and 33, and then slowly increased in number again until day 36. CFC-F were present at day 10, and their numbers increased exponentially until day 15, decreased dramatically until day 24, and then remained constant till day 35. Thus, the transient growth of CFC-F preceded the appearance and growth of CFU-S in the ossicle by two or three days.

Effect of mouse type I interferon on mouse bone marrow cells and peritoneal exudate cells cultured in vitro.

Type I mouse interferon significantly reduces macrophage colony formation from mouse bone marrow cells or mouse peritoneal exudate cells when cultured in vitro in the presence of colony-stimulating factors (CSF) derived from either pregnant mouse uterus (CSFPMUE or LCM (CSFLCM) (concanavalin-A purified preparation from endotoxin-shocked mouse long-conditioned medium). The effect of interferon on granulocyte (G-CFU) or granulocyte-macrophage-mixed (GM-CFU) colonies depended on the CSF used. G-CFU and GM-CFU were somewhat elevated in interferon-treated cells stimulated by CSFPMUE whereas G-CFU and GM-CFU were slightly inhibited in interferon-treated cells stimulated by CSFLCM. The most dramatic effect of interferon was observed in the macrophage population. We conclude that interferon affects differentiation preferentially at the macrophage progenitor cell level.

Effect of azimexon (BM 12.531) on mouse granulocyte-macrophage and monocyte-macrophage progenitor cells.

Treatment of mice with 25 mg/kg azimexon (BM 12.531) resulted in an increase ln granulocyte-macrophage colony-forming cells (GM-CFC) in spleen and bone marrow after a transient depression in the cell populations. Bone marrow monocyte-macrophage colony-forming cells (MM-CFC) increased at 7 days after treatment, and splenic MM CFC were least affected by azimexon treatment. The increase in granulocytic and monocytic colony-forming cells may play a role in the previously reported protection by azimexon against radiation and drug-induced toxicity.

Population sizes of granulocyte-macrophage and monocyte-macrophage colony-forming cells in Sl/Sld.

The sizes of granulocyte-macrophage (CFU-c) and monocyte-macrophage (CFC) progenitor cell populations were measured and compared in Sl/Sld and +/+ mice. In the marrow, the CFU-c and CFC population sizes were 40% and 67%, respectively, of the +/+ marrow CFU-c and CFC population sizes. There was no difference in the size of these two-cell populations in the spleens or thymi of Sl/Sld mice as compared with +/+ mice. Furthermore, the marrow CFC population was found to be heterogeneous by velocity sedimentation. One of the marrow CFC subpopulations is characterized by a velocity sedimentation value of 5.05 mm/h while the other has a value of 6.30 mm/h.