Closed-cycle noble gas recycling system for high-repetition rate high-harmonic generation.

We present a compact closed-loop recycling system for noble and inert gases. It has been developed for an extreme-ultraviolet (XUV) frequency comb based on high-harmonic generation at 100 MHz repetition rate. The system collects gas injected at several bars of backing pressure through a micrometer-sized nozzle into the laser-interaction region with a differential pumping system comprising turbomolecular pumps, and subsequently compresses the gas to a pressure of up to 200 bar. By drastically reducing the waste of expensive gases such as xenon and krypton, it enables the long operation times needed for spectroscopic measurements, as well as for continuous operation of the XUV frequency comb.

Photoelectron tomography with an intra-cavity velocity-map imaging spectrometer at 100 MHz repetition rate.

We present a compact velocity-map imaging (VMI) spectrometer for photoelectron imaging at 100 MHz repetition rate. Ultrashort pulses from a near-infrared frequency comb laser are amplified in a polarization-insensitive passive femtosecond enhancement cavity. In the focus, multi-photon ionization (MPI) of gas-phase atoms is studied tomographically by rotating the laser polarization. We demonstrate the functioning of the VMI spectrometer by reconstructing photoelectron angular momentum distributions from xenon MPI. Our intra-cavity VMI setup collects electron energy spectra at high rates, with the advantage of transferring the coherence of the cavity-stabilized femtosecond pulses to the electrons. In addition, the setup will allow studies of strong-field effects in nanometric tips.

Student attitudes toward lesbian, gay, and bisexual issues: analysis of self-talk categories.

The purpose of this study was to (a) examine the utility of using the thought-listing technique to examine participants' attitudes toward lesbian, gay, and bisexual issues and (b) determine the effectiveness of two new training interventions (rational and experiential) that were designed using cognitive-experiential self-theory (Epstein, 1994). Fifty participants were randomly assigned to one of three treatment conditions (rational training, experiential training, control group). Participants completed a thought-listing technique before and after receiving one of the theoretically based training interventions. Three judges free sorted the 2,481 collected thoughts and identified and defined 25 thought categories. Three trained judges then placed 2,426 (98%) of the thoughts into these categories. Results indicated that different categories of responses (cognitive, affective, behavioral) emerged among the individuals which provided four distinct profiles of how people thought about the topic of homosexuality. Furthermore, chi-square analyses revealed that the experiential group had changes in their thoughts after receiving the workshop. Findings are discussed and suggestions for future research are provided.

Genetic approaches to neurotrauma research: opportunities and potential pitfalls of murine models.

Genetic strategies provide new ways to define the molecular cascades that regulate the responses of the mammalian nervous system to injury. Genetic interventions also provide opportunities to manipulate and control key molecular steps in these cascades, so as to modify the outcome of CNS injury. Most current genetic strategies involve the use of mice, an animal that has not heretofore been used extensively for neurotrauma research. Therefore, one purpose of the present review is to consider how mice respond to neural trauma, focusing especially on recent information that reveals important differences between mice and rats, and between different inbred strains of mice. The second aim of this review is to provide a brief introduction to the opportunities, caveats, and potential pitfalls of studies that use genetically modified animals for neurotrauma research.

The unique histopathological responses of the injured spinal cord. Implications for neuroprotective therapy.

Tissue destruction at the primary site of a spinal cord injury leads to persistent necrosis that progressively enlarges the lesion. Steroids attenuate this necrotizing process and promote tissue repair even though such anti-inflammatory drugs interfere with wound healing in non-CNS organs. To address this paradox, the spinal cord of rats and mice was crushed extradurally and the effects of the following anti-inflammatory agents studied by light microscopical image analysis: allopurinol, aminoguanidine, indomethacin, a bacterial lipopolysaccharide, naproxen, and pregnenolone. The contribution of Wallerian degeneration to progressive necrosis was studied in a mutant mouse strain (WldS) that is characterized by delayed Wallerian degeneration. In rats, the anti-inflammatory agents selectively attenuated progressive necrosis and encouraged wound healing. In mice, considerable tissue repair occurred without pharmacological intervention; this wound-healing process was delayed in the mutant WldS strain. Since spinal cord injury results in concomitant tissue necrosis and wound healing, a goal of neuroprotective therapy is to regulate the dynamic balance between these destructive and reparative processes.

Mechanisms of motor recovery after subtotal spinal cord injury: insights from the study of mice carrying a mutation (WldS) that delays cellular responses to injury.

UNLABELLED: Partial lesions of the mammalian spinal cord result in an immediate motor impairment that recovers gradually over time; however, the cellular mechanisms responsible for the transient nature of this paralysis have not been defined. A unique opportunity to identify those injury-induced cellular responses that mediate the recovery of function has arisen from the discovery of a unique mutant strain of mice in which the onset of Wallerian degeneration is dramatically delayed. In this strain of mice (designated WldS for Wallerian degeneration, slow), many of the cellular responses to spinal cord injury are also delayed. We have used this experimental animal model to evaluate possible causal relationships between these delayed cellular responses and the onset of functional recovery. For this purpose, we have compared the time course of locomotor recovery in C57BL/6 (control) mice and in WldS (mutant) mice by hemisecting the spinal cord at T8 and evaluating locomotor function at daily postoperative intervals. The time course of locomotor recovery (as determined by the Tarlov open-field walking procedure) was substantially delayed in mice carrying the WldS mutation: C57BL/6 control mice began to stand and walk within 6 days (mean Tarlov score of 4), whereas mutant mice did not exhibit comparable locomotor function until 16 days postoperatively. INTERPRETATION AND CONCLUSION: (a) The rapid return of locomotor function in the C57BL/6 mice suggests that the recovery resulted from processes of functional plasticity rather than from regeneration or collateral sprouting of nerve fibers. (b) The marked delay in the return of locomotor function in WldS mice indicates that the processes of neuroplasticity are induced by degenerative changes in the damaged neurons. (c) These strains of mice can be effectively used in future studies to elucidate the specific biochemical and physiological alterations responsible for inducing functional plasticity and restoring locomotor function after spinal cord injury.

Experimental spinal cord injury: Wallerian degeneration in the dorsal column is followed by revascularization, glial proliferation, and nerve regeneration.

The presence of adequate blood supply is a critical factor in recovery from traumatic injuries. We have examined whether the revascularization of the injured tissues is as crucial a precondition for wound healing in the spinal cord as in other organs. The development of the initial primary lesion (PL) after spinal crush injury in rats is followed by the formation of a unique tunnel-like dorsal column lesion (DCL) that extends rostrocaudally for many millimeters from the primary injury site. The DCL has been shown to result from Wallerian degeneration of the long spinal tracts in the dorsal column. In this study, we compared the processes of revascularization, wound healing, and nerve regeneration in the PL and the DCL by light microscopy after a crush injury of the cord. The spinal cord of 54 adults rats was crushed at T8 with jewelers forceps. The rats were allowed to survive from 3 h up to 8 weeks after spinal cord injury. The PL appeared immediately after injury and the DCL began to develop 6 h later. Infiltration of neutrophils, which is the first sign of the inflammatory responses to injury, began several hours later in the DCL than in the PL. Secondary vascular injury then occurred which resulted in hemorrhage around the DCL and rapid enlargement of the lesion during the remainder of the first week. Subsequent changes in the PL and DCL were entirely different. The PL underwent progressive enlargement and cavitation such that by 8 weeks, the lesion contained only very few cells, vessels, and axons scattered between huge fluid-filled cavities. The DCL, on the other hand, was maximal in size at 1 week and declined significantly in size and cavitation thereafter. By 8 weeks it was highly vascularized, contained abundant nerve fibers, and lacked any trace of cavitation. These findings amplify the current view that ischemia plays a critical role in spinal cord trauma by showing that revascularization precedes tissue repair and nerve regeneration in the dorsal columns. We conclude (a) that a well-vascularized lesion permits the ingrowth of glial and other cells which give rise to a supportive matrix for the nerve regeneration and (b) that procedures which induce revascularization or angiogenesis will ameliorate the cascade of progressive tissue necrosis.

Experimental analysis of progressive necrosis after spinal cord trauma in the rat: etiological role of the inflammatory response.

Progressive tissue necrosis is a unique reaction to spinal cord trauma in which the site of injury is gradually transformed into a large, cavity-filled lesion. The earliest histopathological changes after injury include a widely disseminated extravasation of erythrocytes and neutrophils. To test whether such an inflammatory reaction might initiate progressive necrosis, we examined the effects of the following anti-inflammatory treatments: allopurinol (Ap) to inhibit injury-induced xanthine oxidase, indomethacin (I) or naproxen to inhibit constitutive and inducible cyclooxygenase, aminoguanidine (Ag) to inhibit inducible nitric oxide synthase, pregnenolone (P) as a precursor steroid, and a bacterial lipopolysaccharide (L) to stimulate secretory activities of glial cells and macrophages. The spinal cord of adult rats was crushed at T8 with jeweler's forceps and, after 3 or 21 days of treatment, the cords were studied quantitatively by light microscopical image analysis. Ag, Ag+I, or Ap significantly reduced the size of the primary lesion at 3 days postoperatively, while P+L+I did so only after 21 days of treatment. A secondary lesion developed in the dorsal column and gradually extended for many millimeters rostral and caudal from the primary lesion. The size of the dorsal column lesion was diminished by 3-day treatment with Ap and by 21-day treatment with Ap or P+L+I, but Ag or Ag+I had no effect. We conclude that (a) progressive necrosis is initiated and maintained by inflammatory mechanisms and (b) for this reason, treatment with specific anti-inflammatory agents selectively attenuates various components of the necrotizing process.

Genetic influences on cellular reactions to spinal cord injury: activation of macrophages/microglia and astrocytes is delayed in mice carrying a mutation (WldS) that causes delayed Wallerian degeneration.

Reactive changes in macrophages/microglia and astrocytes were evaluated following spinal cord injury in normal mice of the C57BL/6J strain and in mice carrying a mutation (WldS) which delays the onset of Wallerian degeneration in damaged axons. Crush injuries were produced at the T8 level by using an extradural approach; animals were allowed to survive for 2 days to 12 weeks, and spinal cords were prepared for immunocytochemistry using antibodies against Mac1 and glial fibrillary acidid protein (GFAP). In normal mice, Mac1-positive macrophages accumulated at the injury site by 4 days and immunostaining of these cells peaked at 6-8 days. Cells in the gray matter near the crush site and in the ascending dorsal column also exhibited increased Mac1 staining that was prominent at 1 week and remained high at 2-4 weeks. In mice carrying the WldS mutation, the accumulation of macrophages at the injury site and the increase in immunostaining of these cells were delayed, as were the increases in immunostaining in the gray matter and dorsal columns. Both normal and mutant mice exhibited pronounced increases in glial fibrillary acidic protein immunostaining at the edge of the crush site and for some distance both rostral and caudal to the injury; increased immunostaining was also prominent along the ascending dorsal columns. The center of the crush site, which contained connective tissue, remained completely unstained for GFAP. In normal mice, immunostaining for GFAP reached a peak at 1 week postinjury and then declined. In mice carrying the WldS mutation, increases in GFAP immunostaining did not reach a peak until 2-3 weeks postinjury. These results indicate that activation of macrophages, microglia, and astrocytes is delayed and prolonged in mice carrying the WldS mutation.

Genetic influences on cellular reactions to spinal cord injury: a wound-healing response present in normal mice is impaired in mice carrying a mutation (WldS) that causes delayed Wallerian degeneration.

Progressive tissue necrosis is a process unique to the injured mammalian spinal cord which often leads to gradually increasing cavitation and enlargement of the lesion. To evaluate the role of neuronal degeneration in initiating this response, histopathological changes were compared in C57BL and WldS (delayed Wallerian degeneration mutation) mice. The spinal cord was crushed at T8, producing a primary lesion at the site of the trauma and a secondary lesion extending rostrocaudally in the dorsal columns (where long ascending and descending fiber tracts undergo Wallerian degeneration). Cavitation was relatively mild at both sites and developed mainly at the margins of the lesions. In striking contrast to spinal cord injury in rats, progressive necrosis did not occur in mice; instead, the primary and secondary lesion sites became filled in by macrophages and fibroblasts embedded in a well-vascularized collagenous stroma. Quantitative image analysis revealed that the primary lesion decreased dramatically in size and cavitation between 2 and 3 weeks in C57BL, whereas in WldS the reduction in size and cavitation began later (at 4 weeks) and was less complete. The initial development of the secondary lesion began later and its healing was less complete in WldS than C57BL. These results are consistent with the hypothesis that neuronal damage, including Wallerian degeneration, triggers inflammatory responses leading to tissue repair. For this reason, any delay in neuronal degeneration, as in the WldS mutation, results in deficient tissue repair as reflected in the larger size of both primary and dorsal column lesions.

Key role for pregnenolone in combination therapy that promotes recovery after spinal cord injury.

Controlled compressive injury to rat spinal cord was chosen to test therapies that might attenuate the progression of tissue destruction and locomotor deficits that characteristically occur after spinal injury. A highly significant reduction of damage was achieved by immediate postinjury treatment with a combination of the following: an antiinflammatory substance, indomethacin; a stimulator of cytokine secretion, bacterial lipopolysaccharide; and the parent steroid, from which all other steroids arise, pregnenolone. This treatment reduced histopathological changes, spared tissue from secondary injury, and increased restoration of motor function. Remarkably, 11 of 16 of the animals treated with the above combination were able to stand and walk at 21 days after injury, 4 of them almost normally. The results were far superior to those obtained in controls or in animals to which the substances were given separately or in combination of two. This approach may prove to be applicable to nervous system injury, in general, and to injury in other tissues.

Spinal cord injury in the rat: treatment with bacterial lipopolysaccharide and indomethacin enhances cellular repair and locomotor function.

Trauma to the rat's spinal cord results in a lesion characterized by ingrowth of glial cells, accumulation of macrophages, and the progressive development of necrosis and cavitation. Since, when appropriately activated, both astrocytes and macrophages secrete growth-promoting cytokines, we examined whether treatment with drugs that stimulate the secretory activities of these cells might promote tissue repair and reduce necrosis in the traumatized spinal cord. The spinal cord of rats was crushed extradurally at T8 and the rats were injected intraperitoneally with (i) a lipopolysaccharide (LPS) or ImuVert to activate cytokine secretion, (ii) Indomethacin to reduce necrosis by inhibiting prostaglandin synthesis, (iii) a combination of LPS+Indomethacin, or (iv) vehicle. After 28 days the lesion site was examined quantitatively by light microscopical image analysis. The lesion of vehicle-treated control animals showed large cavities, extensive infiltration by debris-engorged macrophages, and relatively few axons. Treatment with LPS or ImuVert significantly reduced the degree of cavitation and increased the number of cells and axons in the lesion. Treatment with LPS+Indomethacin was significantly more effective than treatment with LPS alone, while treatment with Indomethacin alone was ineffective. To test whether the histopathological differences between treated and control rats might be reflected in functional improvement, rats were subjected to a contusion (weight-drop) injury and their walking ability was quantified by the Tarlov scale for 28 days postoperatively. Treatment with LPS+Indomethacin significantly improved locomotor function of animals subjected to a moderate (1.25 g x 20 cm) injury. We conclude that tissue repair and functional recovery after spinal cord injury are enhanced by combined treatment with agents that promote the secretory activities of the nonneuronal cells and that inhibit prostaglandin synthesis. These results indicate that the search for more effective treatments should include studies on combinations of drugs having different pharmacological specificities.

Quantitative evaluation of axonal regeneration by immunochemical assay for neurofilament protein.

In experiments on nerve regeneration requiring assessment of the rate and extent of axonal outgrowth, the availability of a simple and accurate method of quantification would be extremely useful. We approached this issue by modifying the conventional ELISA procedure so as to provide a sensitive, specific, and quantitative biochemical assay of the phosphorylated neurofilament content of homogenates or sections of nerve tissue. The technique involves four sequential steps: (i) adhesion of fixed or fresh homogenates or tissue sections to wells of microtiter plates, (ii) binding of a monoclonal antibody against phosphorylated neurofilament to the tissue, (iii) secondary binding to the anti-phosphorylated neurofilament of a phosphatase-labeled second antibody (antimouse IgG), and (iv) enzymatic assay of alkaline phosphatase activity using a fluorescent substrate (4-methylumbelliferyl phosphate). The technique is sufficiently sensitive to measure the phosphorylated neurofilament content of a 1:100,000 (w/v) homogenate of brain, spinal cord, or peripheral nerve and of single 10-microns paraffin sections of Bouin-fixed rat spinal cord. To validate the applicability of the procedure to the study of nerve regeneration, the sciatic nerve of adult rats was either crushed (to permit regeneration) or transected and ligated (to preclude regeneration). The animals were autopsied 1 to 16 weeks later, when four segments 3-mm in length taken from regions proximal and distal to the lesion site were assayed for phosphorylated filament content. The temporal course of its disappearance during degeneration and its reappearance during regeneration coincided with the known histologic changes in crushed and transected nerves. These findings demonstrate the validity of using the immunochemical assay for PNF in studies of nerve regeneration in the peripheral nervous system and the potential applicability of this procedure to studies on regeneration in the central nervous system.

Distribution of carbonic anhydrase activity in neurons of the rat.

Certain neurons of dorsal root ganglia (DRG) and some fibers of the sciatic nerve contain histochemically demonstrable carbonic anhydrase activity. Since the distribution of this enzyme throughout the nervous system has not yet been evaluated systematically, we conducted a comprehensive histochemical survey focusing particularly on structures derived from the neural crest and nonneural crest ectoderm. In the peripheral nervous system, we observed carbonic anhydrase activity in some, but not all, neurons of dorsal root, trigeminal, celiac, and myenteric ganglia as well as in glial cells throughout the CNS. Some neurons of the nodose ganglion also showed carbonic anhydrase activity. In all first order sensory ganglia that were studied, the enzyme was found only in large (50 micron or above) and medium (20-50 micron) size neurons; in the case of spinal ganglia, the reactive neurons constituted approximately 30% of the total neuronal population. Of these reactive neurons, 56% were heavily stained and 44% were moderately stained. Several possible roles for neuronal carbonic anhydrase are considered.

Essentiality of a specific cellular terrain for growth of axons into a spinal cord lesion.

To date, there are no reports of growth of significant numbers of axons into or across a lesion of the mammalian spinal cord. However, recent studies showing that CNS axons will grow into PNS environments indicate that comparable growth into spinal cord lesions could be achieved if ischemic necrosis could be prevented and the lesion site repopulated by astrocytes and ependymal cells rather than by the macrophages, lymphocytes, and fibroblasts that generally accumulate at sites of CNS injury. To examine this possibility, we made a laminectomy at T5 in rats and crushed the spinal cord for 2 s with a smooth forceps (leaving the dura mater intact to prevent ingrowth of connective tissue). At 1 week, the lesion was filled with mononuclear cells, degenerating nerve fibers, and capillaries that were oriented parallel to the long axis of the spinal cord. By 2 weeks, longitudinally oriented cords of ependymal cells and astrocytes had migrated into the lesion from the adjacent spinal cord, and similarly oriented nerve fibers had begun to grow into the lesion along these capillaries and cellular cordons. The mononuclear cells had now assumed phagocytic activity and were engorged with myelin and other cellular debris. After 3 weeks, the astrocytes had elaborated thick cell processes. The nerve fibers in the lesion were still oriented longitudinally but had increased in number and were often arranged in small fascicles. These observations provide the first histological evidence of growth of nerve fibers into a lesion of the rat spinal cord. We conclude that the intrinsic regenerative capacity of the spinal cord can be expressed if ischemic necrosis and collagenous scarring are prevented and the spinal cord parenchyma is first reconstructed by its nonneuronal constituents.

Enhancement of axonal growth into a spinal lesion by topical application of triethanolamine and cytosine arabinoside.

We have demonstrated that a brief compression lesion of the rat spinal cord produces axotomy with minimal necrosis or scarring and that axons grow into such a lesion along longitudinally oriented capillaries and similarly oriented cordons of ependymal cells and astrocytes. Inasmuch as extensive, oriented growth of axons into a spinal lesion is never seen after transection, concussion, or other models of spinal cord injury, this new surgical procedure appeared to be applicable to the in vivo testing of pharmacological agents designed to promote neuritic outgrowth. The spinal cord of anesthetized rats was crushed extradurally for 1 s with a smooth jeweler's forceps. After 2 days when edema had subsided, the animals were reoperated. The dura mater was opened, and a polyethylene tube was implanted so that one end was fastened over the injury site and the other end was exteriorized at the back of the neck. The lesion site was superfused with 0.1 ml of control or test solutions four times daily for 2 weeks and then the animals were anesthetized and killed by vascular perfusion with fixative. After decalcification, the vertebral column and spinal cord were embedded in paraffin and stained by several histologic procedures including the protargol silver impregnation method for nerve fibers. Treatment with triethanolamine and cytosine arabinoside, substances which promote neuritogenesis in cultured spinal ganglia of chick embryos, markedly stimulated the growth of axons into the lesion of the rat spinal cord. We conclude (i) that it is possible to pharmacologically enhance the intrinsic growth capacity of CNS neurons and (ii) that brief compression provides a type of injury that is well suited to the evaluation of treatments aimed at promoting axonal regeneration.

Differences between adult and neonatal rats in their astroglial response to spinal injury.

Transection of the thoracic spinal cord in adult rats produces an astroglial reaction at the lesion site which spreads gradually to lumbar segments. We compared the spread of gliosis in cordotomized adult and neonatal rats in order to evaluate whether or not maturity of long spinal tracts is a precondition for the genesis of this histopathological reaction. By this experiment, we sought to determine whether spread of gliosis is induced by degeneration of nerve fibers in ascending and descending pathways or results from some more general reaction to injury. The spinal cords of 40 neonatal and 30 young adult rats were transected at T5, and 4 to 60 days later the cervical, thoracic, and lumbar segments were examined immunocytochemically for glial fibrillary acidic protein. In the neonatal rats, there was a moderate gliosis at the lesion site by 7 days; this reaction intensified somewhat during the next 60 days but always remained confined to the site of injury. In contrast, the lesion site of adult rats showed a much more intense gliosis; in those animals the response was maximal by 14 days and was characterized by a gradient of decreasing glial reactivity both rostrally and caudally from the transection site. These results support the hypothesis that the spread of gliosis from spinal lesions results from degeneration of the long ascending and descending fiber tracts.

Development of embryonic spinal cord transplants in the rat.

Although fetal brain tissue, grafted into the CNS of neonatal and adult animals, has been shown to survive and differentiate, relatively little information has been obtained regarding the development of embryonic spinal cord transplants, especially in the injured host CNS. The survival and differentiation of fetal spinal cord transplants in either intracerebral cavities or the lateral ventricles of the adult rat brain were thus examined with light and electron microscopy. Approximately 90% of the spinal cord implants taken from 12-15-day fetuses persisted in either transplantation site with some surviving for as long as 8 months (latest interval studied). The survival rate was considerably lower (22%), however, with tissues obtained from older fetuses. Within 3 weeks, the transplants obtained from 12-15-day donors had become extensively myelinated and contained many neurons of different sizes, including some clusters of large neurons resembling ventral horn cells of the intact spinal cord. In addition, all of the mature grafts were characterized by multiple myelin-free regions of neuropil, containing many small neurons (20 micron in diameter). [3H]Thymidine labelling of the transplants and intact cords of the surviving littermates of the donor fetuses suggested that these myelin-free areas corresponded to the substantia gelatinosa of the adult spinal cord. In many cases, the transplants were confluent with the host CNS parenchyma without an intervening glial scar. Furthermore, multiple spinal cord transplants, placed into the same lesion site, were often fused, and injection of one of the transplants with horseradish peroxidase demonstrated many retrogradely labelled neurons in the adjacent implant. The results of this study suggest that some topographical features of the normal spinal cord may be represented in mature spinal cord transplants. In addition, these findings establish a basis for future investigations aimed at repair of the injured host spinal cord with homologous fetal tissue.