Roy A. Glover

Innervation of the human knee joint by substance-P fibers

Anterior knee pain is a frequent musculoskeletal complaint affecting all ages, both sexes, athletes, and nonathletes alike. Numerous theories have been proposed regarding its etiology including patellar malalignment, quadriceps insufficiency, subluxation, quadriceps muscle tightness, and chondral defects. However, the mechanism by which these factors produce this pain is not clear. Knowledge of the distribution of nociceptive nerve fibers around the knee would seem to provide insight in treating these painful conditions. Eleven human patellae--eight specimens from patients with degenerative patellofemoral disease and three normals--were evaluated. Immunohistochemical techniques using monoclonal antibody to substance-P were employed to identify nociceptive fibers. Substance-P is a nociceptive neurotransmitter found in afferent nerve fibers. Substance-P fibers were isolated in the retinaculum, fat pad, periosteum, and subchondral plate of patellae affected with degenerative disease. This study demonstrates that selective tracting of nociceptive pain fibers is possible around the knee both in soft tissue and, in some circumstances, bone. The subchondral plate of normal patellae did not demonstrate erosion channels, but those with chondral defects from degenerative disease did. Nociceptive fibers found in these defects may explain the origin of symptoms in some patients. The distribution of substance-P nerve fibers in the soft tissues around the knee suggests that denervation may be the mechanism by which surgical procedures for anterior knee pain produce favorable results.

Substance P Innervation of Lumbar Spine Facet Joints

Sixteen adult human lumbar spine facet joints were harvested from patients undergoing various lumbar spine procedures. Diagnoses included degenerative disc disease, adult spinal deformity, facet joint degenerative arthritis, and degenerative spondylolisthesis. Facet joints were processed for routine hematoxylin and eosin staining. Immunohistochemical analysis was performed using a monoclonal antibody to substance P. All facets grossly exhibited evidence of degenerative disease, including cartilage surface irregularity and fibrillation. Histological examination of facets obtained from patients with degenerative spinal conditions demonstrated erosion channels extending through the subchondral bone and calcified cartilage into the articular cartilage. Immunostaining showed the presence of substance P-positive nerve fibers within these erosion channels, and also within marrow spaces. The presence of substance P nerve fibers within subchondral bone of degenerative lumbar facet joints implicates this type of joint in the etiology of low back pain.

Astroglial uptake is modulated by extracellular K

Abstract— Primary cultures of rat brain astrocytes were used to examine the uptake of the glucose analogue, 2‐deoxy‐d‐glucose (2‐DOG). 2‐DOG competes with glucose for uptake, indicating that both are transported by the same carrier system. Extracellular K+ at 11.9 mM increased the uptake of 2‐DOG at 2‐DOG concentrations greater than 100 μM. Uptake. appears Na+‐dependent only at high concentrations of 2‐DOG. This suggests that the extracellular concentrations of Na+ and K+ may regulate the astrocytic uptake of 2‐DOG.

Genetic prenatal aqueductal stenosis with hydrocephalus in rat

A recessive mutation which arose in Wistar albino rats was variably expressed in the homozygous state as prenatal stenosis of the aqueduct with resultant hydrocephalus. The condition was often compatible with survival to adulthood and with successful reproduction. Mildly sparse hair was the constant gene marker. Eye defects and sometimes foot deformities occurred. The first observable ultrastructural alteration was a disruption of the integrity of the neuroepithelial basal lamina in the cephalic neural tube of affected embryos as early as the 11th fetal day (16–24 somite pairs). The hydrocephalic syndrome closely resembled that produced by giving folic acid analogs to, or producing vitamin B12 deficiency in, pregnant rats in the period including the 11th day. Neither vitamin B12 nor folate, nor certain metabolites closely related to their metabolism, prevented the genes expression. Homozygote mutants mated with homozygote mutants produced 70% hydrocephalic (dome-shaped heads) offspring, but if the mother was heterozygote, there was a “protective” effect and the number of hydrocephalic young was disproportionately smailer.

Neuronal cues regulate uptake in cultured astrocytes

Astrocytes of the vertebrate central nervous system are postulated to nourish neurons4,~0,13, to electrically insulate cortical neuronal columns 15, to cue patterns of developmental migrations of neurons 14, and to regulate the ionic environment of brain 5, 17,~9. Despite persistent investigation, the function or functions of this phylogenetically ubiquitous cell type remain largely hypothetical. Neurons and their attendant glia are considered to be a functional metabolic unit. For this to be the case, at least two criteria must be met: (1) some mechanism must exist for communication of relevant information about the metabolic state of one cell-type to the other; and (2) either cell-type must be able to alter its metabolism in response to cues emitted by the other. Although the spectrum of neuronally emitted cues is large, ideally, such cues should reflect some aspect of neuronal activity. In the present study we have selected extracellular K + and 3 putative neurotransmitters as neuronal cues, since it is known that the cerebral levels of both reflect neuronal activity and that each also affects glial membrane potentiala, 18, oxygen uptake 1 and cyclic nucleotide levels 3,9,11. One manner in which neurons may affect glial metabolism is by inducing alterations in the glial uptake of key metabolites, such as glucose, possibly a rate-limiting step in cerebral glycolysis 16 and selected amino acids, the uptake of which by the glial cells may, in part, reflect transfer from the extracellular space of brain tissue 7,12. Recently, we developed a technique for the cultivation of a purified population of astrocytes, derived from neonatal rat forebrain 2 and in the present study we investigate the effect of neuronal cues (see above) on the astrocytic uptake of the glucose analogue 2-deoxy-D-glucose (2-DOG) and of L-methionine (MET), the precursor amino acid essential for the cerebral synthesis of the universal methyl donor molecule, S-adenosylL-methionine. Under the conditions of the experiment described in Fig. 1, the uptake of 2-DOG remained linear for at least 15 min. To determine the effects of extracellular K + on the uptake of 2-DOG, KC1 replaced NaCI on a molar basis. Plates of confluent cells were incubated with 0.25, 0.50, and 1.0 mM 2-DOG at the indicated concentrations of extra-

Beta-alanine uptake is not a marker for brain astroglia in culture.

The properties of the beta amino acid transport system were examined in cultivated rat brain astrocytes, using the specific probe, beta-alanine. The uptake of beta-alanine is thought to be glial specific. Beta-alanine was not actively transported and the intracellular accumulation was not altered by coincubation with GABA, alanine, glutamate, or methionine. We suggested therefore that beta-alanine is passively taken up by a mechanism distinct from the transport system for GABA.

Nociceptive Role of Substance-P in the Knee Joint of a Patient with Congenital Insensitivity to Pain

This case report describes the immunocytochemical examination of tissue from a 9-year-old black child diagnosed with congenital insensitivity to pain at age 5. A recent fall and resulting patella fracture required surgical treatment. Biopsies of the distal pole and surrounding soft tissue, as well as a sample of his patellofemoral joint fluid, were taken at the time of partial patellectomy and analyzed for substance-P (SP). Morphologic staining of the patella showed a grossly degenerated patellofemoral articular surface. Examination of tissue sections stained either immunocytochemically with diaminobenzidine DAB or by a rhodamine fluorescent labeling technique showed no evidence of SP-positive nerve fibers. Furthermore, only a trace amount of SP (7.29 pg/ml) was detected in a sample of the patients knee joint synovial fluid. This patients absence of pain sensation in conjunction with the absence of SP nerve fibers in stained patella sections and identification of only trace levels of SP in his synovial fluid, further implicates this neuropeptide in nociceptive innervation of diarthrodial joints.

A new cell surface relationship between neuroepithelial cells during rat neural tube development

Electron microscopic examination of the developing neural tube in 11th to 13th day rat fetuses revealed a new cell surface relationship between differentiating neuroepithelial cells. Cytoplasmic projections possessing terminal dilations were observed extending from neuroepithelial cells through cytoplasmic furrows into large coated pits at the surface of adjacent cells. This cell-to-cell relationship provides a mechanism for the internalization of surface molecules and possibly even cytoplasmic constituents. Communication between donor and recipient cells mediated in this way suggests a route for the sharing of macromolecules, including cytoplasmic fragments, which could function as regulators in embryonic development and differentiation.

Potassium modulation of methionine uptake in astrocytes in vitro

Methionine participates in a large variety of metabolic pathways in brain, and its transport may play an important regulatory role. The properties of methionine uptake were examined in a preparation of neonatal rat brain astrocytes. Uptake is linear for 15 minutes, up to 2.5 μM. At steady state conditions, methionine is concentrated 30–50-fold. Measured methionine homoexchange accounts for a significant fraction of uptake at concentrations greater than 10 μM. We recently reported that methionine uptake is decreased by elevations in extracellular K+. Potassium induced efflux cannot account for this apparent effect; and thus for concentrations less than 2.5μM, and for short times of incubation, measured rates of methionine uptake represent unidirectional flux. At extracellular concentrations of K+ equal to 6.9 mM, the apparentVmax of methionine transport is 182 pmol/min/mg protein, and theKm is 1.3 μM. Where K+ is shifted to 11.9 mM, theKm remains unchanged, and theVmax is reduced by half.

Alpha-aminoisobutyric acid uptake in primary cultures of astrocytes

Homotypically pure cultures of rat brain astrocytes were used to examine some aspects of non-neuronal A-system (alanine preferring) amino acid uptake. The Asystem specific probe, alpha-aminoisobutyric acid is transported rapidly, and a steady state distribution ratio of 9–25 is reached after 30 minute incubations. Kinetic estimates derived from uptake progress curves indicated aKm of 1.35 mM and aVmax of 133 nmol/min/mg protein. Uptake is reduced in the absence of either Na+ or K+. Elevations in extracellular K+, a putative metabolic modulator of neuroglia, did not affect uptake.