Elvar Theodorsson

Further studies on galanin-, substance P-, and CGRP-like immunoreactivities in primary sensory neurons and spinal cord: Effects of dorsal rhizotomies and sciatic nerve lesions

The peptides galanin (GAL), substance P (SP), and calcitonin gene-related peptide (CGRP) were analyzed with immunohistochemistry and radioimmunoassay in the spinal cord, dorsal root ganglia, dorsal roots, and sciatic nerve of normal rats and rats subjected to several experimental procedures, including ligation, crush, and/or sectioning of nerves. The results show that peripheral nerve transection induces a dramatic increase in GAL content both in dorsal roots and sciatic nerve, demonstrating that this lesion causes an increased out-transport of the newly synthesized peptide both into the central and peripheral branches of the primary sensory neurons. In contrast evidence was obtained for decreased out-transport of SP and CGRP. The functional significance of these findings remains to be analyzed.

Microwave irradiation increases recovery of neuropeptides from brain tissues

The effect of focused high energy microwave treatment (MW) on brain concentrations and molecular forms of substance P, neurokinin A, neuropeptide Y, neurotensin, galanin and calcitonin gene-related peptide was investigated. Groups of rats were treated as follows: 1) MW, storage for 60 min at 22 degrees C, 2) Decapitation, storage for 60 min at 22 degrees C. 3) Decapitation, storage for 60 min at 22 degrees C, MW treatment, 4) MW, decapitation, storage for 2 min at 22 degrees C and 5) Decapitation, storage for 2 min at 22 degrees C. Peptide concentrations were in all instances highest in the MW sacrificed groups. MW increased the concentration of intact peptides by rapid inhibition of peptidase activity and increase in peptide solubility/extractability.

Neuropeptides in brain: effects of microwave irradiation and decapitation.

Substance P (SP)-, neurokinin A (NKA)-, neurotensin (NT)-, neuropeptide Y (NPY)- and vasoactive intestinal polypeptide (VIP)-like immunoreactivity (Ll) were measured and characterized by specific radioimmunoassays (RIA) and reverse phase high performance liquid chromatography (HPLC) in extracts of rat brain. Concentrations of SP-Ll, NKA-Ll and NT-Ll in brains of decapitated animals were 59, 49 and 64 percent lower compared to those found in animals sacrificed by focused microwave irradiation (MW). In contrast, no difference in brain NPY-Ll and VIP-Ll levels was found between animals killed by MW and decapitation. HPLC chromatograms of SP-, NKA-, NT- and NPY-Ll showed the same immunoreactive components in extracts of brains from both groups of animals. Thus, no additional immunoreactive components were formed by MW compared to those found after decapitation. The present findings may reflect an MW-induced inhibition of peptidase activity or, perhaps, a more efficient extraction of certain neuropeptides following MW treatment. The results imply that the traditional methods of sacrifice may result in the measurement of spuriously low tissue concentrations of some peptides, e.g. tachykinins, in brain.

Neuropepties in the saliva of healthy subjects

Five neuropeptides: Substance P (SP), Neurokinin A (NKA), Calcitonin Gene-Related Peptide (CGRP), Neuropeptide Y (NPY) and Vasoactive Intestinal Polypeptide (VIP), were measured in the saliva of eight subjects. The saliva was collected using different stimulation techniques: whole resting saliva, whole paraffin stimulated saliva, whole citric acid stimulated saliva and parotid saliva of different secretion rates -0.25 mL/min, 0.50 mL/min and 1.00 mL/min, also stimulated by citric acid. The neuropeptides were analysed by radioimmunoassay. The results showed that the concentration of all neuropeptides decreased significantly, two- to four-fold (CGRP up to 16-fold) in whole saliva, when the salivary secretion rates increased six- to eight-fold due to stimulation. However, the amounts of all neuropeptides released over time into the whole saliva increased two- to five-fold (ten-fold for CGRP) as the volumes of saliva increased due to chewing-stimulation as compared to resting saliva or citric acid stimulated saliva. There was also more CGRP in the resting saliva than in the citric acid stimulated saliva. The concentration of CGRP in the parotid saliva decreased three- to ten-fold when the salivary flow increased, whereas the concentration of NKA increased three- to four-fold and that of NPY almost two-fold under the same conditions. The concentrations of SP and VIP did not change in the different flows of parotid saliva. The release of all neuropeptides in the parotid saliva over time showed significant increases (3-14-fold) when the secretion rates increased except CGRP, which showed no changes at all. We concluded that neuropeptides are continuously released into the saliva. Their amounts increase with stimulation, but they are diluted by the increased volume of saliva, and they are also affected by the mode of stimulation-muscular activity leads to a greater release than citric acid stimulation. As the neuropeptides play an important role in the control of salivary secretory mechanisms, their normal occurrence and release are of fundamental importance for the understanding of the function of the salivary glands.

Repeated electroconvulsive stimuli: changes in neuropeptide Y, neurotensin and tachykinin concentrations in time

1. The effects of repeated electroconvulsive stimuli (ECS) on neuropeptide Y (NPY)-, neurokinin A (NKA)-, substance P (SP)- and neurotensin (NT)- like immunoreactivity (-LI) levels in specific rat brain regions were studied in order to establish changes in peptide tissue concentrations in time after the last ECS. 2. The rats were sacrificed 15 minutes, 60 minutes, 1 day, 7 days or 15 days after the sixth sham ECS or ECS, using focused microwave irradiation. 3. Following dissection of the brains, peptides were extracted and measured in extract aliquots by radioimmunoassays. 4. ECS increased NPY-LI in both right and left hippocampus, frontal cortex and occipital cortex at 15 min, 60 min and 1 day after the treatments. Seven days following the last treatment, NPY-LI concentrations were elevated in the left hippocampus and occipital cortex but not in the corresponding right structures. Fifteen days following the last ECS treatment no changes in NPY concentrations were seen. 5. Also NKA-LI was increased in both the right and left hippocampus; the duration of changes was identical to those of NPY-LI. 6. No effects on SP- or NT-LI were found. 7. These results are in line with our hypothesis that one of the ECTs mechanisms of action might involve its effects on NPY and NKA.

Increased concentration of calcitonin gene-related peptide in cerebrospinal fluid of depressed patients. A possible trait marker of major depressive disorder

Cerebrospinal fluid (CSF) was collected under controlled conditions from subjects suffering from major depression (n = 63) or schizophrenia (n = 28) and from healthy controls (n = 20). Following Sep-pak extraction, calcitonin gene-related peptide immunoreactivity (CGRP-LI) was determined by radioimmunoassay in sample aliquots. CGRP-LI concentrations in CSF were increased in the depressed patients compared to the schizophrenic and control subjects (P < 0.001). No CGRP-LI differences were found between the latter two groups. CGRP-LI did not correlate to any of the technical (e.g. storage conditions) or patient (demographic, biochemical, or clinical) variables investigated. In view of the CGRPs discrete distribution and specific effects in brain and the above results, we hypothesize that increased CSF CGRP-LI might be a trait marker of major depression. Regardless of the mechanisms (altered synthesis/release/metabolism in brain or changed fate in CSF) leading to elevated CSF CGRP-LI, the identification of a possible disease trait marker should contribute to the early diagnosis of major depression and identification of family members at risk and may help in differential diagnosis in other disorders with affective symptomatology.

Neuroimmunomodulatory effects of acupuncture in mice

The purpose of this study was to assess the effect of acupuncture on the immunological response. The induction of anti-sheep red blood cells (SRBC) plaque-forming cells (PFC) was used as a measurement of the immune response to treatment. In normal non-immunized mice, enhancement of PFC was seen after a single acupuncture treatment when spleen cells from stimulated mice were cultured with SRBC in vitro. After 3 acupuncture treatments, spleen cells from mice did not show PFC enhancement after treatment with anti-Thy-1.2 antibody and complement, nor after the removal of non-adherent cells. Serum obtained from mice 1 h after acupuncture stimulation enhanced the PFC of normal spleen cells in vitro, but the enhancement was abolished by the addition of propranolol. These results suggest that acupuncture, by activation of the autonomic nervous system, modulates the immune response.

Effects of adjuvant on neuropeptide-like immunoreactivity in experimentally induced temporomandibular arthritis in rats

Substance P (SP)-, neurokinin A (NKA)-, calcitonin gene-related peptide (CGRP)- and neuropeptide Y (NPY)-like immunoreactivities (-LI) were examined in cerebrospinal fluid (CSF), plasma and temporomandibular joint (TMJ) perfusates in rats 1 and 12 h after inoculation at the base of the tail (0.05 ml) or injection into the right TMJ (0.01 ml) of heat-killed Mycobacterium butyricum in paraffin oil. In the rats inoculated at the base of the tail (polyarthritic rats), there was a significant increase of CGRP-LI and NKA-LI. The changes in neuropeptide-LI were not as marked in the CSF of rats injected with adjuvant in one TMJ (monoarthritic rats) as in the polyarthritic group. Instead, the most significant changes in the monoarthritic rats were seen in the perfusates of both TMJs. The increases in SP-, NKA-, CGRP- and NPY-LI were significant for both TMJs and more pronounced than in the polyarthritic rats. The results show that inoculation of adjuvant at the base of the tail induces significant changes of neuropeptide-LI predominantly in CSF, whilst an intra-articular injection induces bilateral changes in neuropeptide-LI in joint perfusate. Therefore, two different neural mechanisms may be involved early in adjuvant-induced poly- and monoarthritis.

The effect on joint fluid concentration of neuropeptide Y by intra-articular injection of glucocorticoid in temporomandibular joint arthritis.

Twenty-two patients (29 joints) with temporomandibular joint (TMJ) arthritis of specific or unspecific nature were given one intra-articular glucocorticoid (GC) injection. The effect on subjective symptoms and clinical signs in the craniomandibular system and on joint aspirate concentration of neuropeptide Y-like immunoreactivity (NPY-LI) was evaluated at follow-up visits 2-3 or 4-6 weeks after treatment. In the patients with specific inflammatory joint disease the treatment resulted in an improvement of symptoms and clinical signs and in a reduction in the TMJ level of NPY-LI 2-3 weeks after treatment. In the patients with unspecific inflammatory joint disease there was also an improvement in the clinical variables and a reduction in the NPY-LI level after 2-3 weeks, but not on a statistically significant level. The results of this study show that intra-articular GC treatment causes a short-term decrease of the TMJ fluid level of NPY-LI in patients with specific inflammatory joint disease, while symptoms and signs improve.

Concurrent analysis of neuropeptides and biogenic amines in brain tissue of rats treated with electroconvulsive stimuli

We developed a method for measuring neuropeptides and monoamines in the same rat brain tissue and applied this method to study the effects of electroconvulsive stimuli (ECS) on these compounds. Rats were treated with repeated ECS or sham ECS. After sacrifice by focused microwave irradiation, brains were dissected and immediately frozen. The tissues were extracted in acetic acid. After lyophilization the samples were reconstituted in phosphate buffer and divided in three fractions: (1) was further purified on a cation-exchange column before catecholamines were measured on a high-performance liquid chromatography (HPLC) system, (2) for measuring serotonin on the HPLC system, (3) for measuring peptide concentrations by specific radioimmunoassays. Confirming our previous findings, ECS significantly increased neuropeptide Y-like immunoreactivity (-LI) in hippocampus and frontal cortex and neurokinin A-LI in the hippocampus, while no changes in substance P- and neurotensin-LI were detected. New findings were a decrease in noradrenaline concentrations in the frontal and occipital cortex and hippocampus, an increase in dopamine concentrations in the frontal and occipital cortex and no serotonin change. In summary, we have developed methods to measure both peptides and monoamines in the same brain tissue specimens, and have shown that ECS leads to changes in both neuropeptides and classical neurotransmitters in distinct brain regions.