Computer-assisted Image Processing Research Articles

Computer-Assisted Analysis of Radial Symmetry in Human Airway Epithelial Cilia: Assessment of Congenital Ciliary Defects in Primary Ciliary Dyskinesia

The ultrastructural analysis of cilia and diagnosis of primary ciliary dyskinesia (PCD) in biopsies of airway epithelium is sometimes confounded by poor sampling, inconsistencies in tissue quality and processing, and other technical problems. Although clinical findings maylead to a presumptive diagnosis, ultrastructural analysis of ciliary axonemes is the standard for confirmation of PCD. The ultrastructural features of the ciliumwhen viewed in crosssection by transmission electron microscopy confer a radial symmetry to the axoneme. Current digital image processing techniques can be applied to such images to reinforce signal, diminish noise, and confirm consistency of position of axonemal structures,a process that can augment ultrastructural analysis for PCD. In this study, computer-assisted digital image processing was used to evaluate cross sections of cilia in airway epithelial biopsies from patients previously diagnosed with PCD as well as in control subjects with normal cilia. These studies supported the original diagnoses and provided some new insights into axonemal organization in PCD. This technique is simple and may be useful in providing a supporting means for confirming or ruling out a diagnosis of PCD in cases that appear equivocal.

Development of a 1-km landcover dataset of China using AVHRR data

This paper describes the development of a 1-km landcover dataset of China by using monthly NDVI data spanning April 1992 through March 1993. The method used combined unsupervised and supervised classification of NDVI data from AVHRR. It is composed of five steps: (a) unsupervised clustering of monthly AVHRR NDVI maximum value composites is performed using the ISOCLASS algorithm; (b) preliminary identification is carried out with the addition of digital elevation models, eco-region data and a collection of other landcover/vegetation reference data to identify the clusters with single landcover classes; (c) re-clustering is performed of clusters with size greater than a given threshold value and containing two or more disparate landcover classes; (d) cluster combining is performed to combine all clusters with a single landcover class in one cluster, and all other clusters into one mixed cluster; and (e) supervised classification is used to carry out post-classification of the mixed cluster generated in the previous step by using the maximum likelihood algorithm and the identified single landcover classes of the previous step as training data. The classification is based on extensive use of computer-assisted image processing and tools, as well as the skills of the human interpreter to take the final decisions regarding the relationship between spectral classes defined using unsupervised methods and landscape characteristics that are used to define landcover classes.

Microvascular endothelium dysfunction in rats bearing 1,2-dimethylhydrazine-induced colon tumors

Functional parameters (adhesivity, permeability, thrombogenity and thromboresistance) of the mesenteric microvasculature endothelium in rats bearing 1,2-dimethylhydrazine (DMH) induced colon tumors were studied using a complex method, based upon computer-assisted microscopic video image processing. It was shown that paraneoplastic endothelial dysfunction in rat mesenteric microvessels is characterized by an increase in leukocyte adhesion to the endothelium, microvessel permeability and thrombogenic properties. It was demonstrated that the observed changes are the consequences of tumor growth and are related to tumor size and the duration of the process.

Virtual reality of the lower urinary tract in women.

Advances in computerized and imaging technology permit both students and doctors to depict the anatomy of the human pelvis more realistically than with previous methods. Further refinements outline fine pelvic structures, such as the nerve plexus, which may as a result be spared during major pelvic surgery, thus preserving the function of the bladder neck and urethra. Dynamic computerized tomography or magnetic resonance imaging, coupled with three-dimensional depiction of the lower urinary tract and its adjacent structures, enable visualization of the whole lower urinary tract and the pelvic floor musculature in both continent and incontinent women. In patients with a reconstructed lower urinary tract computer-assisted image processing shows the postoperatively altered topographical anatomy. This may be clinically useful for interpretation of unexpected findings with conventional imaging modalities, postoperative morbidity, and surgical planning of a lower abdominal reoperation. Examples of our own work regarding the innervation of female pelvic organs, dynamic depiction of the bladder and pelvic floor musculature during straining in normal and incontinent women, and the situation of female patients after undergoing an anterior pelvic exenteration with subsequent orthotopic neobladder procedure, are given. In addition, the data of these patients have been compiled for virtual reality endoscopy, which is useful for patient consent and for teaching residents, students and nurses.

Determination of the Influence of Wind on the Keweenaw Current in the Lake Superior Basin as Identified by Advanced Very High Resolution Radiometer (AVHRR) Imagery

The Keweenaw Current is a warm coastal current in Lake Superior that flows northeastward along the northern shore of Michigan's Keweenaw Peninsula. This study focuses on the fate of the current at the tip of the Keweenaw Peninsula. Results of this study suggest that the path of the current beyond the peninsula is primarily controlled by wind. In this study, eleven surface temperature maps derived from Advanced Very High Resolution Radiometric (AVHRR) data using computer assisted image processing techniques were used to identify the Keweenaw Current. Wind data from two moored data buoys, a Coastal Marine Automated Network (CMAN) fixed buoy, and three airport weather stations, collected on the same day as each of the images and for the two days preceding the image date, are used to determine whether wind direction and speed influence the path of the Keweenaw Current past the tip of the peninsula. In nine images the current's path is similar to the surface Ekman transport direction predicted from wind data. All eleven images show a similarity between the current's actual path and a path calculated when net Ekman transport is assumed. Results of this study also show that there may be a possible lag time of one day between a change in wind direction and the current's adjustment to that change.

Morphometrical comparison of human spermatozoa obtained from semen and swim-up methodology

Because morphology is regularly established in semen smears, but not in swim-up spermatozoa, we were interested in comparing some morphological parameters of semen and swim-up spermatozoa to establish if the cells selected by the swim-up method were morphologically similar to those considered normal in semen. Normal human semen samples were divided into two aliquots. One of these aliquots was washed by centrifugation with B2 medium and sperm smears were prepared with the resulting pellet as a control. The other aliquot was used to perform swim-up separation and the spermatozoa from the supernatant were used as experimental smears. Both groups were stained according to the triple stain technique and spontaneous acrosome reaction and viability were determined. Video microscopy and computer-assisted image processing of live and non-reacted sperm cells were used to establish morphometrical parameters of the sperm head in both populations. The following set of morphometrical parameters were considered: width, length, width/length ratio, acrosome area, head area, and acrosome area/head area ratio. An increase in head width, a decrease in head length and a subsequent increase of width/length ratio were found in swim-up cells compared with the control group. A slight increase in acrosome area/head area ratio was also observed in swim-up supermatozoa. Through the swim-up methodology we were able to select a subpopulation of oval shaped heads with spermatozoa having a bigger acrosome area in comparison to semen.

Succinate dehydrogenase activities of fibers in the rat extensor digitorum longus, soleus, and cardiac muscles.

Succinate dehydrogenase (SDH) activities and cross-sectional areas (CSAs) of different types of fibers in the superficial (EDLs) and deep (EDLd) regions of the extensor digitorum longus and soleus (SOL) muscles and the left ventricular muscle of the heart (HEART) of 10-week-old male rats were determined using quantitative histochemistry and a computer-assisted image processing system. The fibers were classified as type I, type IIA, type IIB, or type IIC according to their histochemically assessed adenosine triphosphatase activities. The mean SDH activity was higher and the mean CSA was smaller in type IIA fibers than in type IIB fibers in both the EDLs and EDLd. The mean SDH activity of type IIA fibers in the SOL was higher than that of type I fibers. Fibers in the HEART showed the highest mean SDH activity and the smallest mean CSA among all fiber types in the muscles examined. There was an inverse correlation between CSA and SDH activity for the different fiber types in different muscles. These data suggest that the SDH activity of fibers in muscle is fiber type- and size-specific, and that the highest SDH activity of fibers in the left ventricular muscle of the heart contributes to their functional properties, i.e., high fatigue resistance.

Hypergravity exposure decreases gamma-aminobutyric acid immunoreactivity in axon terminals contacting pyramidal cells in the rat somatosensory cortex: a quantitative immunocytochemical image analysis.

Quantitative evaluation of gamma-aminobutyric acid immunoreactivity (GABA-IR) in the hindlimb representation of the rat somatosensory cortex after 14 days of exposure to hypergravity (hyper-G) was conducted by using computer-assisted image processing. The area of GABA-IR axosomatic terminals apposed to pyramidal cells of cortical layer V was reduced in rats exposed to hyper-G compared with control rats, which were exposed either to rotation alone or to vivarium conditions. Based on previous immunocytochemical and behavioral studies, we suggest that this reduction is due to changes in sensory feedback information from muscle receptors. Consequently, priorities for muscle recruitment are altered at the cortical level, and a new pattern of muscle activity is thus generated. It is proposed that the reduction observed in GABA-IR of the terminal area around pyramidal neurons is the immunocytochemical expression of changes in the activity of GABAergic cells that participate in reprogramming motor outputs to achieve effective movement control in response to alterations in the afferent information.

Hypergravity exposure decreases γ‐aminobutyric acid immunoreactivity in axon terminals contacting pyramidal cells in the rat somatosensory cortex: A quantitative immunocytochemical image analysis

Quantitative evaluation of γ-aminobutyric acid immunoreactivity (GABA-IR) in the hindlimb representation of the rat somatosensory cortex after 14 days of exposure to hypergravity (hyper-G) was conducted by using computer-assisted image processing. The area of GABA-IR axosomatic terminals apposed to pyramidal cells of cortical layer V was reduced in rats exposed to hyper-G compared with control rats, which were exposed either to rotation alone or to vivarium conditions. Based on previous immunocytochemical and behavioral studies, we suggest that this reduction is due to changes in sensory feedback information from muscle receptors. Consequently, priorities for muscle recruitment are altered at the cortical level, and a new pattern of muscle activity is thus generated. It is proposed that the reduction observed in GABA-IR of the terminal area around pyramidal neurons is the immunocytochemical expression of changes in the activity of GABAergic cells that participate in reprogramming motor outputs to achieve effective movement control in response to alterations in the afferent information. J. Neurosci. Res. 53:135–142, 1998. © 1998 Wiley-Liss, Inc.

An automatic diagnostic system for CT liver image classification.

Computed tomography (CT) images have been widely used for liver disease diagnosis. Designing and developing computer-assisted image processing techniques to help doctors improve their diagnosis has received considerable interests over the past years. In this paper, a CT liver image diagnostic classification system is presented which will automatically find, extract the CT liver boundary and further classify liver diseases. The system comprises a detect-before-extract (DBE) system which automatically finds the liver boundary and a neural network liver classifier which uses specially designed feature descriptors to distinguish normal liver, two types of liver tumors, hepatoma and hemageoma. The DBE system applies the concept of the normalized fractional Brownian motion model to find an initial liver boundary and then uses a deformable contour model to precisely delineate the liver boundary. The neural network is included to classify liver tumors into hepatoma and hemageoma. It is implemented by a modified probabilistic neural network (PNN) [MPNN] in conjunction with feature descriptors which are generated by fractal feature information and the gray-level co-occurrence matrix. The proposed system was evaluated by 30 liver cases and shown to be efficient and very effective.

Confocal Microscopic Characterization of a Lesion in a Cerebral Vessel of the Stroke-Prone Spontaneously Hypertensive Rat

Hypertension is a major risk factor for stroke and is associated with alterations in vascular structure and function. The aim of this study was to determine vascular function, wall morphology, and vascular smooth muscle cell (VSMC) arrangement in basilar arteries from stroke-prone spontaneously hypertensive rats (SHRSP) and normotensive control strain Wistar-Kyoto rats (WKY). The effect of perindopril treatment on SHRSP structure and function was also assessed. VSMC orientation was determined with laser-scanning confocal microscopy and computer-assisted image processing in basilar arteries stained with 5(6)-carboxyfluorescein (wavelengths: excitation, 488; emission, 515) or propidium iodide (excitation, 529; emission, 550). Measurements of wall morphology and functional responses to serotonin and KCl were assessed with wire myography. In the WKY basilar arteries, VSMCs were uniformly oriented perpendicular to the longitudinal axis of the vessel, whereas in the SHRSP there were localized foci of VSMC geometric disorganization, with a significant deviation from 90 degrees. The SHRSP basilar arteries also showed structural remodeling and reduced contractile responses to serotonin and KCl. Perindopril treatment normalized blood pressure, prevented wall morphology alterations, and improved function but had no effect on VSMC disorganization. This is the first demonstration of lesions of VSMC geometric disorganization in a cerebral artery from a stroke-prone genetically hypertensive rat strain. These structural abnormalities are independent of blood pressure. Their functional sequel may play a role in the pathogenesis of stroke in this model.

Motility of rhG-CSF-induced neutrophils in patients undergoing chemotherapy: evidence for inhibition detected by image analysis.

The motility of circulating neutrophils from seven patients affected by intermediate and high-grade non-Hodgkin's lymphoma was investigated before and after rhG-CSF administration (5 micrograms/kg/d for 5 d subcutaneously) in the course of chemotherapy. Random motility and bacterial lipopolysaccharide-induced chemotaxis were studied by the micropore filter technique in a Boyden chamber. These functions were evaluated by a very sensitive technique, based on a computer-assisted image processing system, capable of giving several parameters about the kinetics of cell migration. Along with a significant increase in neutrophil number, a significant decrease both in random and stimulated motility was found. The kinetics of cell migration showed that the cells maintained the typical gaussian pattern of random motility. On the contrary, neutrophils were found to have lost the typical stimulated migration peak. These findings are consistent with a rhG-CSF-induced impairment of the directional movement, rather than of the ability of moving at random. These effects were found in patients who, in the same experimental conditions, had displayed an enhanced phagocytosis and phagocytosis-associated chemiluminescence along with an enhanced CD32 expression, not due to an aspecific cell manipulation. Two hypotheses may be taken into account: (i) an increased adhesiveness due to a direct or an indirect activity of the cytokine; (ii) an abnormality in the cytoskeleton maturation and/or rearrangement during the accelerated bone marrow transit of myeloid cells. These findings emphasize that rh-GCSF administration can modulate several functions which play an important role in host defence, and suggest the utility of carrying out further studies to investigate the optimum dosage both to correct neutrophil number and preserve neutrophil functional activities.

Expression of heat-shock/stress proteins in Duchenne muscular dystrophy.

Heat-shock/stress proteins (HSPs) are induced in response to stressful conditions and are essential for survival during and after cellular stress. We investigated whether dystrophin deficiency in muscle from Duchenne muscular dystrophy (DMD) patients induces HSPs. Immunohistochemical studies were performed on cryosections from normal muscle, heat-shocked muscle, and muscle from patients with DMD, dermatomyositis, and mitochondrial myopathy using antibodies against HSP 72/73, HSP 72, HSP 90, groEL (HSP 65 homologue), and ubiquitin. Computer-assisted image processing revealed a significant (P < 0.05) induction of HSP 72/73, 72, 65, and ubiquitin in hypercontracted fibers; HSP 90 and ubiquitin in regenerating fibers; and ubiquitin in macrophage invaded necrotic fibers of DMD muscle. No significant induction of HSPs was observed in dermatomyositis or mitochondrial myopathies. The stress response induced in DMD may relate to the metabolic stress characteristic of the disease and could represent an autoprotective mechanism. Manipulation of this protective response may reduce injury and have potential therapeutic application.

Impairment of Afferent Arteriolar Myogenic Responsiveness in the Galactose-Fed Rat

Previous studies from our laboratory have demonstrated impaired afferent arteriolar responsiveness to pressure in rats 4-6 weeks after the induction of diabetes mellitus. Although the responsible mechanisms mediating this renal autoregulatory defect have not been fully defined, increased polyol metabolism has been implicated as a possible factor involved in the pathogenesis of diabetic complications. We therefore investigated the possible role of this metabolic disturbance in renal autoregulation using the galactose-fed rat, a model characterized by increased polyol pathway activity independent of hyperglycemia or insulin deficiency. Hydronephrosis was induced to permit direct visualization of renal microvessels. Pressure-induced vasoconstriction of afferent arterioles was assessed by quantitating vessel diameter following stepwise increments of renal perfusion pressure (RAP; from 80 to 180 mm Hg) in the hydronephrotic kidneys from control rats and rats fed a 50% galactose diet for 2 or 4 weeks. Vessel diameters were measured from video images by computer-assisted image processing. Control rats exhibited progressive afferent arteriolar vasoconstriction when RAP was increased from 80 to 180 mm Hg (-17.3% +/- 1.0%; P < 0.001). In contrast, myogenic responses to increases in pressure were absent in the afferent arterioles of rats fed a 50% galactose diet for either 2 (-4.1% +/- 1.9%; not significant) or 4 weeks (-2.9 +/- 3.4%; not significant). Our demonstration that the impairment of afferent arteriolar responsiveness to increasing RAP in the normoglycemic galactose-fed rat was identical to that observed in the STZ-diabetic rat suggests that increased polyol accumulation may contribute to the impairment of renal autoregulation in the diabetic rat.

Altered tachykinin expression by dorsal root ganglion neurons in a rat model of neuropathic pain

The experiments described in the present study approached nerve injury from both a biochemical and anatomical perspective by monitoring changes in expression of preprotachykinin (PPT) mRNA encoding the prototypic tachykinin substance P and related peptide species in neurons of the rat dorsal root ganglia (DRG) following unilateral chronic constriction injury of the sciatic nerve. In situ hybridization histochemistry (ISHH) analyses in conjunction with computer-assisted image processing were employed to quantify levels of PPT mRNA distributed in DRG neurons. Injury-induced changes in PPT mRNA expression by affected DRG neurons included: 1. (1) at early postoperative times, generally increased levels of PPT mRNA associated with small and intermediate-size B cells exhibiting normal morphology, 2. (2) at late postoperative times, markedly decreased levels of PPT mRNA associated with degenerating B cells, and 3. (3) induction of PPT gene expression by large A cells which is highly correlated with degenerative morphological changes. The significant aspects of these changes are discussed with special emphasis on the contribution of altered transmitter expression by DRG neurons to the pathophysiology of causalgia. In particular, the induction of PPT gene expression by many of the large neurons undergoing degenerative changes may represent an important biochemical parameter which is associated with the development and persistence of experimental allodynia.

The antioxidant butylated hydroxytoluene prevents early cholesterol-induced microcirculatory changes in rabbits.

Microcirculation was studied during 10 wk in untreated rabbits (n = 13) and in rabbits treated with dietary addition of 1% cholesterol (n = 13), 1% cholesterol + 1% of the antioxidant BHT (butylated hydroxytoluene) (n = 11), or 1% BHT (n = 5). The studies were performed by direct intravital microscopic imaging of the left and right conjunctivae with the use of a stereo microscope and a high resolution television camera. Microvessel diameter, erythrocyte flow velocity, and microhemorheologic conditions were evaluated quantitatively via a computer-assisted digital image processing system. Significant and marked changes occurred in all the above variables as a consequence of cholesterol feeding. After 3 wk of feeding there was a dramatic decrease (approximately 30%) in blood flow velocity in arterioli of the third order (P < 0.0001), accompanied by aggregation of cells in 40-50% of the smaller conjunctival vessels (P < 0.0001). These changes were enhanced further during the following 7 wk of treatment. All the above changes in the microcirculation were markedly reduced by the addition of BHT treatment. The diameter of the above arterioli decreased in the purely cholesterol-fed group (P < 0.005), whereas this did not occur in the group fed both cholesterol and BHT. In rabbits fed BHT in the absence of cholesterol, there was no significant effect on any assessed microcirculatory variable. In conclusion, the results demonstrate that the antioxidant BHT prevented early cholesterol-induced microcirculatory changes. This prevention occurred in the absence of a reduction of blood lipid levels. The results provide strong support for the hypothesis that a considerable part of the effects on microcirculation in hypercholesterolemia may be due to cholesterol-induced oxidations and not to cholesterol itself. The results are discussed in relation to the previously demonstrated antiatherogenic effect of BHT and the possible use of antioxidants in the therapy and prophylaxis of atherosclerosis.

The effects of chronic haloperidol administration on GABA‐immunoreactive. Axon terminals in rat medial prefrontal cortex

Several reports have suggested that chronic haloperidol (HAL) treatment induces ultrastructural changes in synapses of substantia nigra, corpus striatum, and medial prefrontal cortex (mPFC) of rat brain. The effects of HAL on specific cortical transmitter systems, however, are not well characterized. Recent studies have indicated that there may be a loss of gamma-aminobutyric acid (GABA)ergic cells in anterior cingulate cortex of schizophrenic subjects and this hypothesis has prompted interest in the question of whether dopamine receptor antagonists, such as HAL, may influence the activity of this transmitter system. This current report describes a quantitative light microscopic analysis of GABA-immunolabeled axosomatic terminals in mPFC of rats treated with HAL decanoate (0.5 mg/kg/day, i.m.) for a period of 4 months. GABA-containing terminals were visualized with an avidin-biotin immunoperoxidase method for localizing anti-GABA antibodies. Computer-assisted image processing was employed to determine the total number of pixels representing GABA-immunoreaction product in axon terminals that were in direct apposition to pyramidal cell bodies. Drug-treated animals showed a significant increase in the number of pixels representing GABA-immunoreaction product in axosomatic terminals of layers II, III, VI, and VI (93%, 63%, 31%, and 43%, respectively). These data are consistent with the idea that chronic HAL administration may be associated with a significant increase in the amount of GABA present in terminals surrounding pyramidal neurons of rat mPFC. The fact that GABA-containing terminals showed the greatest increase in layer II is not consistent with the known distribution of dopamine afferents to this region which is lowest in superficial laminae. Based on the laminar distribution of non-dopaminergic receptor types that have a high affinity for HAL, the effect of this drug on GABAergic transmission could potentially involve changes that are mediated through mechanisms in which 5-HT2 or sigma opiate receptors play a role.

Changes of spinal substance P, calcitonin gene-related peptide, somatostatin, Met-enkephalin and neurotensin in rats in response to formalin-induced pain

Changes of substance P (SP)-, calcitonin gene-related peptide (CGRP)-, somatostatin (SS)-, Met-enkephalin (Met-Enk)- and neurotensin (NT)- immunoreactive materials on two sides of the lumbar dorsal horn were inspected microscopically and quantified with a computer-assisted image processing system in rats with intact or totally transected spinal cord 2 h after injection of 0.2 ml of 0.5% formalin into the right hindpaw subcutaneously. The results showed that the SP-like immunoreactivity (SP-LI), CGRP-LI, SS-LI, Met-Enk-LI, and NT-LI were significantly higher in fibers and terminals in superficial laminae of the dorsal horn ipsilateral to the formalin injection in both of the experimental groups. It is supposed that the increased contents of these peptides reflect an increased biosynthesis, transport, and release of these peptides in primary afferents and spinal intrinsic neurons in response to the long-lasting inflow of noxious messages, and that these changes seem to be produced even in the condition when the supraspinal effects have been excluded.

Confocal microscopy and computer-assisted image reconstruction of astrocytes in the mammalian retina.

The distribution of astrocytes in the vascularized retina of pigs, rats and cats was investigated by confocal microscopy and computer-assisted image processing. In whole mounts, immunocytochemical identification was done by staining astrocytes for glial fibrillary acidic protein (GFAP), and blood vessels for alpha-smooth muscle actin or collagen IV. Double-staining was followed through consecutive optical sections and made it possible to precisely align the two markers in the inner retina. The resulting computer-assisted image reconstructions revealed asymmetric ensheathment of blood vessels by GFAP-positive fibres. The ultrastructural basis for this asymmetry, as studied by electron microscopy, was found to be different in pigs and cats. In the pig, astrocytes firmly ensheathed the vessel circumference, but glial filaments were much more abundant on the vitreal and lateral than on the scleral side. By contrast, in the cat astrocytes were generally confined to regions occupied by axonal bundles and constituted only part of the vascular glia limitans, else formed by Müller cells. Moreover, our observations unambiguously showed that individual astrocytes maintained simultaneous contact with axons and blood vessels and lined the vitreous body. The physical links provided by astrocytes suggest that they are able to function as central communicating elements between ganglion cells, the vasculature and the vitreous body.

Spatial patterns of increased spinal cord membrane-bound protein kinase C and their relation to increases in 14C-2-deoxyglucose metabolic activity in rats with painful peripheral mononeuropathy.

1. Three-dimensional spatial patterns of changes in membrane-bound protein kinase C (PKC) were examined in the lumbar spinal cords (L1-L5) of rats with an experimental painful peripheral mononeuropathy. Painful peripheral mononeuropathy was produced by loosely ligating the rat's common sciatic nerve, resulting in chronic constrictive nerve injury (CCI). Changes in spinal cord membrane-bound PKC distribution were assayed by employing an established quantitative [3H]-phorbol-12,13-dibutyrate ([3H]PDBu) autoradiographic assay, which includes spinal cord sectioning, incubation of spinal cord sections with [3H]PDBu, production of autoradiographs, and computer-assisted image processing. 2. Sciatic nerve ligation induced demonstrable thermal hyperalgesia in response to radiant heat stimulation and spontaneous pain-related behaviors (such as lifting of the nerve-ligated hind paw) in CCI rats 3, 7, and 10 days after unilateral sciatic nerve ligation. 3. Consistent with behavioral changes, CCI rats examined 3 or 10 days after sciatic nerve ligation displayed a three-dimensional pattern of increased membrane-bound PKC in the lumbar spinal cord (L1-L5) strikingly different from that of sham-operated rats: in the dorsoventral dimension, reliable increases in membrane-bound PKC occurred mainly within spinal cord laminae I-IV and V-VI in CCI rats; in the ipsilateral-contralateral dimension, changes in membrane-bound PKC were seen on both sides of the spinal cord in CCI rats with reliably higher levels of membrane-bound PKC on the side ipsilateral than on the side contralateral to sciatic nerve ligation; in the rostrocaudal dimension, increases in membrane-bound PKC in the spinal cord dorsal horns of CCI rats extended from spinal segments L2-L5. 4. Both three-dimensional increases in spinal cord membrane-bound PKC and nociceptive behaviors (thermal hyperalgesia and spontaneous pain behaviors) in CCI rats were reliably reduced after three daily intrathecal treatments with 80 nmol GM1 ganglioside (a glycosphingolipid shown to prevent PKC translocation/activation), the first of which was given 1 h after sciatic nerve ligation. This reduction was seen 24 h but not 7 days after the last GM1 ganglioside treatment. 5. This three-dimensional increase in membrane-bound PKC in the spinal cord dorsal horn of CCI rats displayed high correlations with thermal hyperalgesia and with spontaneous pain-related behaviors in CCI rats observed both 3 and 10 days after sciatic nerve ligation. Similar correlations were observed between decreases in levels of membrane-bound PKC in the spinal cord dorsal horn and the attenuation of nociceptive behaviors in CCI rats after three daily intrathecal treatments with GM1 ganglioside.(ABSTRACT TRUNCATED AT 400 WORDS)