Karin Larsson

Phosphate‐deficient oat replaces a major portion of the plasma membrane phospholipids with the galactolipid digalactosyldiacylglycerol

The plasma membranes of oat normally resemble those of other eukaryotes in containing mainly phospholipids and sterols. We here report the novel finding that the galactolipid digalactosyldiacylglycerol (DGDG) can constitute a substantial proportion of oat plasma membrane lipids, in both shoots and roots. When oat was cultivated under severe phosphate limitation, up to 70% of the plasma membrane phosphoglycerolipids were replaced by DGDG. Our finding not only reflects a far more developed potential for plasticity in plasma membrane lipid composition than often assumed, but also merits interest in the context of the limited phosphate availability in many soils.

Ultrastructural changes in spinal nerve roots induced by autologous nucleus pulposus

Study Design Ultrastructural changes were analyzed by transmission electron microscopy in nerve roots exposed to autologous nucleus pulposus experimentally. Objectives To assess if ultrastructural changes were present in areas with no light microscopic changes in nerve roots exposed to autologous nucleus pulposus in a pig model. Summary of Background Data Previous analyses have shown that there is focal nerve fiber damage in nerve roots exposed to autologous nucleus pulposus in the pig. These changes could not fully explain the reduction in nerve conduction velocity seen in the same nerve roots. In the present study, the parts of the nerve roots that did not display breakdown of axons or myelin sheaths at the light microscopic level were analyzed regarding ultrastructural changes. Methods In a previous study, nucleus pulposus was harvested from a lumbar disc and placed epidurally onto the cauda equina at the sacrococcygeal level in pigs. Retroperitoneal fat was used as control. After 1, 3, and 7 days, the nerve roots were excised and processed for light microscopy. Parts of the nerve roots that appeared normal at the light microscopic level were further processed for the present electron microscopic examination. Results Significant ultrastructural changes, such as expansion of the Schwann cell cytoplasm and intracellular edema with vesicular swelling of the Schmidt‐Lanterman incisures, were observed in nerve fibers with normal axons. Although present after nucleus pulposus and control application, the changes were more pronounced after the application of nucleus pulposus. Conclusions Epidural application of autologous nucleus pulposus without any pressure may induce not only nerve function impairment but also axonal injury and significant primary Schwann cell damage with vesicular swelling of the Schmidt‐Lanterman incisures. However, because axonal and Schwann cell changes affected only part of the nerve fibers, further causes of the impaired nerve conduction need to be determined.

Phosphate-limited Oat THE PLASMA MEMBRANE AND THE TONOPLAST AS MAJOR TARGETS FOR PHOSPHOLIPID-TO-GLYCOLIPID REPLACEMENT AND STIMULATION OF PHOSPHOLIPASES IN THE PLASMA MEMBRANE

We recently reported that cultivation of oat (Avena sativa L.) without phosphate resulted in plasma membrane phosphoglycerolipids being replaced to a large extent by digalactosyldiacylglycerol (DGDG) (Andersson, M. X., Stridh, M. H., Larsson, K. E., Liljenberg, C., and Sandelius, A. S. (2003) FEBS Lett. 537, 128–132). We report here that DGDG is not the only non-phosphorous-containing lipid that replaces phospholipids but that also the content of glucosylceramides and sterolglycosides increased in plasma membranes as a response to phosphate starvation. In addition, phosphate deficiency induced similar changes in lipid composition in the tonoplast. The phospholipid-to-glycolipid replacement apparently did not occur to any greater extent in endoplasmic reticulum, Golgi apparatus, or mitochondrial inner membranes. In contrast to the marked effects on lipid composition, the polypeptide patterns were largely similar between root plasma membranes from well-fertilized and phosphate-limited oat, although the latter condition induced at least four polypeptides, including a chaperone of the HSP80 or HSP90 family, a phosphate transporter, and a bacterial-type phosphoesterase. The latter polypeptide reacted with an antibody raised against a phosphate deficiency-induced phospholipase C from Arabidopsis thaliana (Nakamura, Y., Awai, K., Masuda, T., Yoshioka, Y., Takamiya, K., and Ohta, H. (2005) J. Biol. Chem. 280, 7469–7476). In plasma membranes from oat, however, a phospholipase D-type activity and a phosphatidic acid phosphatase were the dominant lipase activities induced by phosphate deficiency. Our results reflect a highly developed plasticity in the lipid composition of the plasma membrane and the tonoplast. In addition, phosphate deficiency-induced alterations in plasma membrane lipid composition may involve different sets of lipid-metabolizing enzymes in different plant tissues or species, at different stages of plant development and/or at different stages of stress adjustments.

Proinflammatory cytokines in cerebrospinal fluid and serum in patients with disc herniation and sciatica.

Abstract. Proinflammatory cytokines have been identified in herniated intervertebral discs in humans, and such cytokines have experimentally been demonstrated to be important in the pathophysiological mechanisms of disc herniation. Cerebrospinal fluid (CSF) and serum concentrations of interleukin (IL)-1β, IL-6, IL-8, interferon (IFN)-γ and tumour necrosis factor (TNF)-α were investigated using the enzyme-linked immunosorbent assay (ELISA) technique in 39 patients with lumbar disc herniation and sciatica. Pain duration and pain intensity (visual analogue scale, VAS) were recorded at inclusion, and a clinical examination was performed evaluating neurological findings. The extent of disc herniation (protrusion or extrusion/sequestration) was evaluated perioperatively. Normal concentrations of IL-1β, IL-6, IFN-γ and TNF-α were present in CSF and serum in almost all patients with lumbar disc herniation. The concentrations of IL-8 in CSF were increased in 12 out of 39 patients, and these increased levels of IL-8 correlated to a short duration of pain and to more pronounced herniation (extrusion or sequestration). No relationship between IL-8 concentrations in CSF and pain intensity, positive neurological findings or a positive straight leg-raising (SLR) test was found. The observation of increased concentrations of IL-8 in CSF in patients with a short duration of symptoms supports the concept of the initial involvement of inflammatory mechanisms after a disc herniation. The finding that most of the patients with increased concentrations of IL-8 in CSF had an extrusion or a sequestration may suggest that the increase in IL-8 is related to mechanical nerve root compression, but may also indicate a biochemical effect exerted by the herniated disc on the surrounding tissue. Further studies on the potential role of IL-8 as a biomarker for disc herniation are warranted.

Membrane phospholipids as a phosphate reserve: the dynamic nature of phospholipid‐to‐digalactosyl diacylglycerol exchange in higher plants

It is well established that phosphate deficiency induces the replacement of membrane phospholipid with non-phosphorous lipids in extra-plastidial membranes (e.g. plasma membrane, tonoplast, mitochondria). The predominant replacement lipid is digalactosyl diacylglycerol (DGDG). This paper reports that the phospholipid-to-DGDG replacement is reversible, and that when oat seedlings are re-supplied with radio-labelled phosphate, it is initially recovered primarily in phosphatidylcholine (PC). Within 2 d, the shoot contains more than half of the lipid-associated radiolabel, reflecting phosphate translocation. Oat was also cultivated in different concentrations of phosphate and the DGDG/PC ratio in roots and phospholipase activities in isolated plasma membranes was assayed after different times of cultivation. The DGDG/PC ratio in root tissue correlated more closely with plasma membrane-localized phospholipase D, yielding phosphatidic acid (PA), than with plasma membrane-localized PA phosphatase, the activity that results in a decreased proportion of phospolipids. The lipid degradation data did not reflect a significant involvement of phospholipase C, although a putative phospholipase C analogue, non-specific phospholipase C4 (NPC4), was present in oat roots. The correlation between increased phospholipase D activity and DGDG/PC ratio is consistent with a model where phospholipid-to-DGDG replacement involves formation of PA that readily is removed from the plasma membrane for further degradation elsewhere.

Cultured, autologous nucleus pulposus cells induce functional changes in spinal nerve roots.

Study Design. Nerve conduction velocity in pig nerve roots was assessed after application of various preparations of nucleus pulposus and control. Objective. To study whether cultured nucleus pulposus cells could reduce nerve conduction velocity after epidural application. Summary of Background Data. It is known that nucleus pulposus applied epidurally may reduce the nerve conduction velocity of the adjacent nerve roots and that this reduction seems to be related to the cells of the nucleus pulposus. Methods. Nucleus pulposus cells and fibroblasts were cultured for 3 weeks, and various preparations were applied to the cauda equina in 29 pigs. The cells were always from the same animals from which they had been harvested. After 1 week, nerve conduction velocity was determined by local electrical stimulation. Results. Application of live fibroblasts and conditioned culture medium from the nucleus pulposus cell culture dishes did not induce significant reduction of conduction velocity, compared with application of dead fibroblasts, which served as control. However, application of live and dead nucleus pulposus cells induced significant reductions. Conclusions. Application of nucleus pulposus cells reproduced the previously seen reduction in nerve conduction velocity induced by nonmodified nucleus pulposus. Because membranes of the nucleus pulposus cells had similar effects, it can be assumed that the effects are related to membrane‐bound substances or structures.

A rapid transport route between the epidural space and the intraneural capillaries of the nerve roots.

Study Design The possibility of epidurally applied substances reaching the intraneural capillaries of the spinal nerve roots and cauda equina was assessed in the pig sacrococcygeal spine. Methods The presence of Evans blue-labelled albumin in intraneural capillaries after epidural applicaton for 1, 10, or 30 minutes was studied with fluorescence microscopy. Ink angiography was used to determine whether there were any direct communicating vessels between the epidural vien plexus and the intraneural capillaries. Results Evans blue-labelled albumin was present in the intraneural capillaries 1 minute after epidural application. Microangiography demonstrated small venules that connected the epidural vein plexus and the intraneural capillaries. Conclusions The results of this study demonstrated a rapid transport route between the epidural space and the intraneural capillaries. The results suggest that nucleus pulposus material, as well as epidurally applied substances, such as local anesthetic drugs or epidurally injected corticosteroids, may have a rapid, direct transport route to the axons of the spinal nerve roots. The demonstrated transport route also may be related to the mechanisms behind epidural anesthesia and spinal nerve root infiltration.

Infliximab Attenuates Immunoreactivity of Brain-derived Neurotrophic Factor in a Rat Model of Herniated Nucleus Pulposus

Study Design. The effect of infliximab, a chimeric monoclonal antibody to TNF-&agr;, on induction of brain-derived neurotrophic factor (BDNF) was examined using an experimental herniated nucleus pulposus (NP) model. Objectives. To investigate whether treatment of infliximab could attenuate an induction of BDNF, which functions as a modulator of pain, following NP application to the nerve root. Summary of Background Data. Evidence from basic scientific studies proposes that TNF-&agr; is involved in the development of NP-induced nerve injuries. However, the therapeutic mechanisms of infliximab against pain have not been elucidated experimentally. Methods. Twenty rats were used in this study. In the test groups, the animals underwent application of NP to the L4 nerve roots and received a single systemic (intraperitoneal) injection of infliximab at the time of surgery (Infli-0 group, n = 5) or at 1 day after operation (Infli-1 group, n = 5). As a control treatment, sterile water was administered intraperitoneally to 5 rats with NP application (NP group) and to 5 sham-operated rats (sham group). On day 3 after surgery, the L4 dorsal root ganglion (DRG) and L4 spinal segment were harvested and assessed regarding BDNF immunoreactivity. Results. Application of NP induced a marked increase of BDNF immunoreactivity in number in the DRG neurons and within the superficial layer in the dorsal horn compared with the sham group (P < 0.01). Infliximab treatment in the Infli-0 and Infli-1 groups reduced the BDNF induction in both DRG and spinal cord (P < 0.05). Conclusion. These findings indicate that infliximab attenuates the elevated BDNF levels induced by NP. The present study therefore further indicates the importance of TNF-&agr; in sciatica due to disc herniation and the possible therapeutic use of a TNF-&agr; inhibitor for this condition.

Autoimmune properties of nucleus pulposus: an experimental study in pigs.

Study Design. Assessment of activated T and B cells in a subcutaneous chamber filled with autologous nucleus pulposus using flow cytometry and immunohistochemistry. Objectives. To examine if subcutaneously placed autologous nucleus pulposus may attract activated T and B cells in an animal model. Summary of Background Data. Nucleus pulposus has been suggested to trigger an autoimmune response if exposed to the immune system, for example, in association with disc herniation. T-cell activation represents a hallmark in the generation of an autoimmune response, subsequently leading to the differentiation of B cells, but a causal association between the exposure of nucleus pulposus to the systemic circulation and T and B cell activation is still lacking. Methods. Autologous nucleus pulposus was harvested from the intervertebral disc of 9 pigs and placed subcutaneously in perforated titanium chambers. In order to control for the effect of the titanium chamber, an additional empty chamber was placed subcutaneously in each pig. After 7 days, the pigs were killed and the chambers were harvested. Flow cytometry and immunohistochemistry were used for analysis of T-helper cells (CD4+), cytotoxic T cells (CD8+), and B cells (Ig&kgr;) in the chamber exudates and T cells (CD45RC) in the remaining blood clot tissue of the chamber. Results. As compared with the empty chambers, the proportion of activated T cells (CD4+ and CD8+) was significantly higher in the exudate of the nucleus pulposus filled chamber. The proportion of activated B cells expressing immunoglobulin kappa (Ig&kgr;) was also significantly elevated in the exudate of the nucleus pulposus chambers. The analysis of the remaining chamber tissue revealed a significantly higher amount of T cells (CD45RC) in the nucleus pulposus chambers than in the empty chambers. Conclusions. The present findings indicate that nucleus pulposus attracts activated T and B cells. However, since the cell population in the nucleus pulposus of young pigs may differ from that of adult humans, the obtained data may not be directly transferred to the human situation of a disc herniation. The observations in the present study may nevertheless explain some of the local tissue reactions occurring in association with disc herniation and nerve root involvement, thereby providing further insight into the pathophysiology of sciatica.

Walking analysis of rats subjected to experimental disc herniation

Abstract In an attempt to evaluate whether experimental disc herniation can result in changes in walking pattern, presumably indicating pain, four groups of rats – sham (n = 5), disc puncture (NP, n = 5), displacement of nerve root and ganglion (DIS, n = 5), and the combination of disc puncture and displacement (NP + DIS, n = 6) – were assessed when walking in a Plexiglas corridor at day 2–14 after surgery. All surgical procedures were performed at the L4–5 level on the left side. Step length analysis showed that rats in the NP and DIS series had no difference between the legs initially, but a tendency towards slightly shorter left steps at day 8–12, whereas the animals in the NP + DIS series had slightly shorter right steps day 2–10. However, no statistically significant differences compared with the sham series could be detected for any of the groups. Nerve dysfunction on the operated side was only observed on one occasion in two rats and on 2 days in one rat, all from the NP + DIS series. Apparent limping was seen in three of the animals in the NP + DIS series and in one in the DIS series. Limping and nerve dysfunction only co-incided on one occasion out of a total of 13 observations of limping, suggesting that the limping was induced by pain and not neurologic deficit. In conclusion, the combination of epidural nucleus pulposus and displacement induced a limping whereas nucleus pulposus or displacment alone did not. Assessment of limping thus seemed to provide adequate information of presence of pain, but step length measurement did not provide any useful data. Although walking analysis may be a valuable assessment of sciatic pain, better modalities must be developed to analyze experimentally induced nerve root pain.