Chronic Injury Group Research Articles

HLA-DR Overexpression in Tubules of Renal Allografts During Early and Late Renal Allograft Injuries

We sought to discover which types of injuries were related to human leukocyte antigen DR expression in acute rejection and late chronic injury in renal allografts. Ninety-two recipients were separated into the early acute rejection group, the late monocyte infiltration group, and the late chronic injury group. Ten subjects with acute cellular rejection received a repeat biopsy. All samples were stained with CD4, CD8, CD20, CD68, and human leukocyte antigen DR by immunochemical staining. Levels of these markers were compared among the subgroups of each group. Human leukocyte antigen DR expression was greater in the early C4d-negative acute rejection group than it was in the early C4d-positive acute rejection group. Human leukocyte antigen DR expression was greater during acute rejection than that was on a repeat biopsy. Human leukocyte antigen DR expression was accord with the infiltration of monocyte infiltration in the acute cellular rejection group. Human leukocyte antigen DR expression was greater during late acute rejection than it was in BK virus nephropathy, which was not in accord with monocyte infiltration. Human leukocyte antigen DR expression was greater during chronic rejection than it was in IgAN, BK virus nephropathy, and TA/IF groups, and even in tubular atrophy. Human leukocyte antigen DR expression in renal tubular cells was associated with early acute cellular rejection and was in accord with monocyte infiltration. Human leukocyte antigen DR expression in renal tubular cells during the late phase (especially in tubular atrophy) was a marker of chronic rejection, but was not in accord with monocyte infiltration in renal allografts.

Neurologic injury because of trauma after Type II odontoid nonunion

Background contextTreatment of Type II odontoid fractures remains controversial, whereas nonoperative treatment is well accepted for isolated Type III odontoid fractures. Little is known about long-term sequelae of nonoperative management or risk of recurrent injury after nonsurgical treatment. We hypothesize that a substantial proportion of odontoid fractures assumed to be acute are actually chronic injuries and have a high rate of late displacement resulting in neurologic injury. PurposeTo identify patients presenting with previously unrecognized odontoid fracture nonunions and to document the incidence of new neurologic injury after secondary trauma in this population. Study designRetrospective case series. Patient sampleOne hundred thirty-three patients with Type II odontoid fractures presenting to a Level I trauma center. Outcome measuresComputed tomography (CT) and magnetic resonance imaging (MRI) scans, American Spinal Injury Association Motor Score (AMS), and neurologic examination. MethodsAll patients presenting after traumatic injury to a Level I trauma center from May 2005 to May 2010 with a Type II odontoid fracture on CT scan were included. Patients aged less than 18 years and those with pathologic fractures were excluded. Fractures were classified as chronic or acute based on CT evidence of chronic injury/nonunion including fracture resorption, sclerosis, and cyst formation. Magnetic resonance imaging was then examined for evidence of fracture acuity (increased signal in C2 on T2 images). Patients without evidence of acute fracture on MRI were considered to have chronic injuries. Computed tomography and MRI scans were interpreted independently by two reviewers. Chart review was performed to document demographics, AMS, and new-onset neurologic deficit associated with secondary injury. ResultsOne hundred thirty-three patients presented with Type II odontoid fractures and no known history of cervical fracture with an average age of 79 years. Based on CT criteria, 31/133 (23%) fractures were chronic injuries. Nine additional fractures appeared acute on CT but were determined to be chronic by MRI findings. The overall number of chronic fractures was therefore 40 (30%). Interobserver reliability analysis for classification of fractures as chronic demonstrated κ=0.65 representing substantial agreement. Of the 40 chronic fractures, 7 patients (17.5%) had new-onset neurologic deficits after secondary injury including 4 motor deficits, 2 sensory deficits, and 1 combined deficit. Although the chronic injury group as a whole had similar AMS to the acute injury group (89 vs. 84, p=.27), the seven patients with new-onset neurologic deficit had an average AMS of 52.4. ConclusionsA substantial proportion of patients presenting after cervical trauma with Type II odontoid fractures have evidence of nonacute injury. Of these patients, 17% presented with a new neurologic deficit caused by an “acute-on-chronic” injury.

Detection of ADAMTS-4 Activity Using a Fluorogenic Peptide-Conjugated Au Nanoparticle Probe in Human Knee Synovial Fluid

A disintegrin and metalloproteinase with thrombospondin motif-4 (ADAMTS-4) plays a pivotal role in degrading aggrecan, which is an early event in cartilage degrading joint diseases such as osteoarthritis (OA). Detection of ADAMTS-4 activity could provide useful clinical information for early diagnosis of such diseases and disease-modifying therapy. Therefore, we developed a ADAMTS-4 detective fluorescent turn-on AuNP probe (ADAMTS-4-D-Au probe) by conjugating gold nanoparticles with a FITC-modified ADAMTS-4-specific peptide (DVQEFRGVTAVIR). When the ADAMTS-4-D-Au probe was incubated with ADAMTS-4, the fluorescence recovered and fluorescence intensity markedly increased in proportion to concentrations of ADAMTS-4 and the probe. A nearly 3-fold increase in fluorescent intensity in response to only 3.9 pM of ADAMTS-4 was detected, whereas almost no fluorescence recovery was observed when the probe was incubated with matrix metalloproteinase (MMP)-1, -3, and -13. These results indicate a relative high sensitivity and specificity of the probe. Moreover, ADAMTS-4-D-Au probe was used to detect ADAMTS-4 activity in synovial fluid from 11 knee surgery patients. A substantial increase in fluorescent intensity was observed in the acute joint injury group as compared to the chronic joint injury and end-stage OA groups, indicating that this simple and low-cost sensing system might serve as a new detection method for ADAMTS-4 activity in biological samples and in screens for inhibitors for ADAMTS-4-related joint diseases. Additionally, this probe could be a potential biomarker for early diagnosis of cartilage-degrading joint diseases.

Effects of Sulfotanshinone Sodium Injection on neuropathic pain in rats

Objective To investigate the effects of Sulfotanshinone Sodium Injection (SSI) on neuropathic pain in rats.Methods One hundred and eight adult male Wistar rats,aged 6-8 weeks,weighing 180-220 g,were randomly divided into 3 groups (n =36 each):sham operation group (group S) ; chronic constrictive injury (CCI)group; group SSI.The animals were anesthetized with intraperitoneal 10% chloral hydrate 350 mg/kg.In groups CCI and SSI,4 ligatures were placed on the right sciatic nerve at 1 mm intervals with 4-0 silk thread according to the method described by Bennett et al.In group S,the right sciatic nerves were exposed,but not ligated.In group SSI,SSI 25 mg/kg was injected intraperitoneally once a day starting from the end of operation until one day before the animals were sacrificed,while the rats received the equal volume of normal saline (5 ml/kg) instead of SSI in groups S and CCI.Twelve animals in each group were chosen at 1 day before operation and 3,7 and 14 days after CCI (T1-4) to measure mechanical paw withdrawal threshold to yon Frey stimuli (MWT) and paw withdrawal latency to thermal nociceptive stimulus (TWL).Six rats in each group were sacrificed at T2-4 after measurement of pain threshold,and their lumbar segnents (L4-6) of the spinal cord were immediately removed for determination of Bcl2 and caspase-3 expression in spinal dorsal horn (by immune-histochemistry),and MDA content and SOD activity (by spectrophotometry) in spinal cord.Results Compared with group S,PWT was significantly decreased,PWL was shortened,the expression of Bcl-2 and caspase-3 was up-regulated,MDA content was increased and SOD activity was decreased at T2-4 in groups CCI and SSI (P ＜ 0.05).Compared with group CNP,PWT was significantly increased,PWL was prolonged,the expression of Bcl-2 was up-regulated,the expression of caspase-3 was downregulated,MDA content was decreased and SOD activity was increased at T2-4 in group SSI (P ＜ 0.05).Conclusion SSI can mitigate neuropathic pain in rats and inhibition of oxidative stress in spinal cord tissues and reduction of apoptosis in spinal dorsal horn neurons are involved in the mechanism. Key words: TANSHINONE ; Neuralgia

Role of ERK signaling pathway in spinal cord in reduction of neuropathic pain by electro-acupuncture at Zusanli and Yanglingquan

Objective To investigate the role of ERK signaling pathway in spinal cord in reduction of neuropathic pain (NP) by electro-acupuncture (EA) at Zusanli and Yanglingquan.Methods Fifty male SpragueDawley rats,weighing 180-220 g,in which the intrathecal catheter was successfully placed without complications,were randomly divided into 5 groups (n =10 each):sham operation group (S group),chronic constrictive injury (CCI) group,acupuncture at acupoint group (EA group),acupuncture at non-acupoint group (NA-EA group),and ERK inhibitor U0126 group (U0126 group).NP was induced by CCI in groups CCI,EA,NA-EA and U0126.The animals were anesthetized with intraperitoneal chloral hydrate 300 mg/kg.The right sciatic nerve was exposed and 4 ligatures were placed on the sciatic nerve at 1 mm intervals.EA (intensity ＜ 1.5 mA,frequency 2Hz) of Zusanli and Yanglingquan lasting 30 min was performed once a day for 6 days starting from 4th day after induction of NP in group EA.EA was performed at the points 5 mm lateral to the acupoints of Zusanli and Yanglingquan on the operated side in group NA-EA.Intrathecal U0126 5 μg was started on 4th day after induction of NP twice a day for 6 consecutive days.The mechanical withdrawal threshold (MWT) and thermal withdrawal latency (TWL) were measured at 1 day before CCI (T0,baseline) and at days 3,5,7 and 9 after CCI (T1-4).The rats were sacrificed on 10th day after induction of NP and the right lumbar segment (L4-6) of the spinal cord was removed for determination of the expression of P2X3 receptor protein and mRNA,ERK1/2 and phosphorylated ERK1/2 (p-ERK1/2) in the spinal cord dorsal horn.Results Compared with group S,MWT was significantly decreased,TWL was significantly shortened,the expression of P2X3 receptor protein and mRNA,and p-ERK1/2 was up-regulated in the other 4 groups (P ＜ 0.01).Compared with CCI group,MWT was significantly increased,TWL was significantly prolenged,the expression of P2X3 receptor protein and mRNA,and p-ERK1/2 was downregulated in EA and U0126 groups (P ＜ 0.01),and no significant change in the parameters mentioned above was found in NA-EA group (P ＞ 0.05).Compared with EA group,MWT was significantly increased,TWL was significantly prolonged,the expression of P2X3 receptor protein and mRNA,and p-ERK1/2 was down-regulated in U0126 group (P ＜ 0.01).Conclusion ERK signaling pathway in spinal cord is involved in reduction of NP by EA at Zusanli and Yanglingquan. Key words: Mitogen-activated protein kinases; Electric stimulation therapy; POINT ST36 (ZUSANLI); POINT GB34 (YANGLINGQUAN); Neuralgia

Effect of curcumin on expression of cannabinoid receptor 1 and NR2B in spinal cord dorsal horn and dorsal root ganglion neurons in a rat model of neuropathic pain

Objective To investigate the effect of curcumin on the expression of cannabinoid receptor 1 (CBR1) and NR2B subunit-containing NMDA receptor in spinal cord dorsal horn and dorsal root ganglion (DRG) neurons in a rat model of neuropathic pain.Methods Seventy-two male Sprague-Dawley rats,weighing 200-230 g,were randomly divided into 4 groups ( n =18 each):sham operation group (S group),chronic constrictive injury (CCI) group,curcumin group (Cur group) and solvent control group (SC group).Neuropathic pain was induced by CCI.The sciatic nerve was exposed and 4 loose ligatures were placed on the sciatic nerve at 1 mm intervals in groups CCI,Cur and SC.Curcumin was injected intraperitoneally at 100 mg· kg- 1· d-1 for 14 consecutive days after operation in Cur group,while the equal volume of dimethyl sulfoxide was given instead of curcumin in SC group.Mechanical withdrawal threshold (MWT) and thermal withdrawal latency (TWL) were measured on 2 days before operation and on 1,3,7,10 and 14 days after operation.Six rats in each group were sacrificed on 3,7 and 14 days after operation and the lumbar segments of the spinal cord ( L4,5 ) and DRGs were removed to determine the expression of CBR1 and NR2B by immuno-histochemistry.Results Compared with S group,MWT was significantly decreased,TWL was significantly shortened,and the expression of CBR1 and NR2B in spinal cord dorsal horn and DRG neurons was up-regulated after operation in the other three groups (P ＜ 0.05 ).Compared with CCI group,MWT was significantly increased,TWL was significantly prolonged,the expression of CBR1 inspinal cord dorsal horn and DRG neurons was up-regulated,and the expression of NR2B in spinal cord dorsal horn and DRG neurons was down-regulated after operation in group Cur (P ＜ 0.05 ),and no significant change in the parameters mentioned above was found in group SC ( P ＞ 0.05 ).Conclusion Curcumin can alleviate neuropathic pain in rats,and up-regulation of CBR1 expression and down-regulation of NR2B expression in spinal cord dorsal horn and DRG neurons are involved in the mechanism. Key words: Curcumin; Neuralgia; Receptors, cannabinoid, CB1; Receptors, N-methyl-D-aspartate; Spinal cord; Ganglia, spinal

Effects of dexmedetomidine on neuronal apoptosis in spinal dorsal horn in a rat model of chronic neuropathic pain

Objective To investigate the effects of dexmedetomidine (Dex) on the neuronal apoptosis in spinal dorsal horn in a rat model of chronic neuropathic pain.Methods Seventy-two adult male SD rats weighing 180-220 g were randomly divided into 3 groups ( n =24 each):sham operation group (group S) ; chronic constrictive injury (CCI) group; Dex + CCI group (group D).Two ligatures were placed on right sciatic nerve at 1 mm intervals with 4-0 silk thread in groups CCI and D.In group D Dex 50 μg/kg was injected intraperitoneally once a day starting from the end of operation until the animals were sacrificed.Paw withdrawal threshold to mechanical stimulation with yon Frey filament (PWT) and paw withdrawal latency to thermal stimulation (PWL) were measured at one day before (T0,baseline) and on the 3rd,7th and 14th day after operation (T1,2,3).Six animals were sacrificed at each time points (T1,2,3) after the measurement of PWT and PWL.Their lumbar segments (L4,5)were removed for examination with transmission electron microscope and detection of Bcl-2 and caspase-3 expression (by immune-histochemistry).Results CCI significantly decreased PWT and PWL,increased Bcl-2 and caspase-3 expression at T1,2,3 and induced apoptosis in spinal dorsal horn neurons in group CCI as compared with group S.Intraperitoneal Dex significantly attenuated CCI-induced mechanical and thermal hyperalgesia and neuronal apoptosis in group D as compared with group CCI.Dex injected intraperitoneally further increased Bcl-2 expression but decreased caspase-3 expression in group D as compared with group CCI.Conclusion Reduction in neuronal apoptosis in spinal dorsal horn is involved in the attenuation of neuropathic pain by Dex. Key words: Dexmedetomidine; Apoptosis; Posterior horn cells; Neuralgia

Role of Akt/GSK-3β signaling pathway in neuropathic pain in mice

Objective To investigate the role of Akt/glycogen synthase kinase-3β (GSK-3β) signaling pathway in neuropathic pain in mice.Methods Thirty-six male C57BL/6 mice,aged 7-8 months,weighing 20-25 g,were randomly divided into 2 groups:sham operation group (group.S,n =9) and chronic constrictive injury (CCI) group (n =27).CCI was produced by placing loosely constrictive ligatures around the common sciatic nerve.Six animals were taken from each group and paw withdrawal threshold to mechanical stimulation (PWMT)and paw withdrawal latency to thermal stimuli (PWTL) were measured on day 1 before operation and on day 1,3,5,7,10,14,17 and 21 after CCI.Three mice were sacrificed on day 3 after CCI in group S and on day 1,3,5,7,10,14 and 21 after CCI in group CCI.The L3-5 segment of the spinal cord was removed for determination of the expression of Akt,phospho-Akt and phospho-GSK-3β by Western blot.Results Compared with group S,PWMT was significantly decreased and PWTL was significantly shortened on day 1-21 after CCI,the expression of Akt was significantly up-regulated on day 7-21 after CCI,and the expression of phospho-Akt and phospho-GSK-3βwas significantly up-regulated on day 1-21 after CCI in group CCI ( P ＜ 0.05).Conclusion Akt/GSK-3β signaling pathway is involved in the development and maintenance of neuropathic pain in mice. Key words: Protein-serine-yhreonine kinases; Glycogen synthase kinase 3; Neuralgia

Evaluation of in Vivo Rotator Cuff Muscle Function After Acute and Chronic Detachment of the Supraspinatus Tendon

Surgical repair of large chronic rotator cuff tears can be technically demanding because it requires manipulation of a muscle-tendon unit that is scarred, retracted, and stiffer than normal, all of which contribute to increased tension at the repair site. The purpose of the present study was to characterize the in vivo rotator cuff muscle-tendon unit function after acute and chronic injury at surgically relevant preload tensions. Sixty-two Sprague-Dawley rats were divided into a healthy, uninjured (control) group (n = 22), an acute injury group (n = 20), and a chronic injury group (n = 20) and underwent in vivo muscle force testing and electromyographic testing of the supraspinatus muscle-tendon unit at various preload tensions. Preload tension affected the maximum supraspinatus muscle contractile force in all groups (p < 0.05). At the peak tension required to repair an acute tear, there was a 28% to 30% reduction in maximum tetanic contraction amplitude in all groups (p < 0.05). At the peak tension required to repair a chronic tear, there was a 40% to 53% reduction in maximal tetanic contraction amplitude in all groups (p < 0.05). The uninjured (control) group showed increased muscle endurance (p < 0.05) in comparison with the acute injury and chronic injury groups at all preload tensions. The chronic injury group showed reduced compound motor action potential amplitude (p < 0.05). Both the acute and chronic injury groups demonstrated functional impairment related to increasing preload tensions. Higher repair tensions, associated with the chronic injury setting, resulted in greater functional impairment. The present study also demonstrates an association between increased time from rotator cuff tendon injury and impaired in vivo rotator cuff muscle electromyographic findings.

Expression changes of parvalbumin and microtubule-associated protein 2 induced by chronic constriction injury in rat dorsal root ganglia.

Parvalbumin (PV), as a mobile endogenous calcium buffer, plays an important role in affecting temporospatial characteristics of calcium transients and in modulating calcium homeostasis. PV is expressed in neurons in the dorsal root ganglion (DRG) and spinal dorsal horn and may be involved in synaptic transmission through regulating cytoplasm calcium concentrations. But the exact role of PV in peripheral sensory neurons remains unknown. Microtubule-associated protein 2 (MAP-2), belonging to structural microtubule-associated protein family, is especially vulnerable to acute central nervous system (CNS) injury, and there will be rapid loss of MAP-2 at the injury site. The present study investigated the changes of PV expressing neurons and the MAP-2 neurons in the DRG after an operation for chronic constriction injury to the unilateral sciatic nerve (CCI-SN), in order to demonstrate the possible roles of PV and MAP-2 in transmission and modulation of peripheral nociceptive information. Seventy-two adult male Sprague-Dawley (SD) rats, weighing 180 - 220 g, were randomly divided into two groups (36 rats in each group), the sham operation group and chronic constriction injury (CCI) group. Six rats in each group were randomly selected to receive mechanical and thermal sensitivity tests at one day before operation and 1, 3, 5, 7, and 14 days after surgery. After pain behavioral test, ipsilateral lumbar fifth DRGs were removed and double immunofluorescence staining was performed to assess the expression changes of PV and of MAP2 expressing neurons in the L5 DRG before or after surgery. The animals with CCI-SN showed obvious mechanical allodynia and thermal hyperalgesia (P < 0.05). Both the thermal and mechanical hyperalgesia decreased to their lowest degree at 7 days after surgery compared to the baseline before surgery (P < 0.01). In normal rats before surgery, a large number of neurons were MAP-2 single labeled cells, and just a small number of PV-expressed neurons were found. PV-positive neurons, PV-positive nerve fibers and PV-negative neurons, formed a direct or close contact for cross-talk. We used immunocytochemical staining to quantify the time course of changes to PV and MAP-2 expressing neurons in tissue, and found that the number of PV expressing neurons began to slightly decrease at 3 days after surgery, and had a significant reduction at CCI day 5, day 7 (P < 0.05). But MAP-2 neurons significantly decreased on just the 3rd day after CCI (P < 0.05). No changes in PV and MAP-2 expression were almost found in sham operated rats. The number of PV positive neurons, was positively correlated with the hyperalgesia threshold. A sharp decline in MAP-2 neurons may be the early response to surgical injury, and PV positive neurons were much more effective at affecting the changes of pain behaviors, indicating that the down-regulation of PV protein could participate in, at least in part, the modulation of nociceptive transmission.

Role of cyclooxygenases in up-regulation of P2X3 receptor expression in dorsal root ganglion in rats with neuropathic pain

Objective To investigate the role of cyclooxygenases (COXs) in the up-regulation of the expression of P2X3 receptors in the dorsal root ganglion (DRG) in rats with neuropsthic pain. Methods Twenty-four male SD rats, weighing 250-280 g, were randomly divided into 4 groups ( n = 6 each): sham operation group (group S), chronic constrictive injury (CCI) group, COX-1 inhibitor ibuprofen group (group Ⅰ), and COX-2 inhibitor celecoxib group (group C). Neuropathic pain was induced by CCI. The animals were anesthetized with intraperitoneal 10% chloral hydrate 300-500 mg/kg. CCI was produced by placing 4 ligatures on the left sciatic nerve at 1 mm intervals. In group S, the left sciatic nerve was only exposed but not ligated. In groups Ⅰ and C, ibuprofen 40 mg·kg-1 ·d-1 and celecoxib 30 mg·kg-1 ·d-1 were given through a gastric tube into the stomach at day 3-14 after operation respectively. Paw withdrawal latency (PWL) and paw withdrawal threshold (PWT) were measured before operation (baseline), and at 3, 5, 7, 10 and 14 days after operation. Then the rats were sacrificed and their L()-6 DRGs were removed to detect the expression of P2X3 mRNA and protein. Results Compared with group S, PWL was significantly shortened, PWT decreased, and P2X3 mRNA and protein expression up-regulated in group CCI ( P ＜ 0.05＝. Compared with group CCI, PWL was significantly prolonged, PWT increased, and P2X3 mRNA and protein expression down-regulated in groups Ⅰ and C (P ＜0.05＝. Compared with group Ⅰ, PWL was significantly prolonged, PWT increased, and P2X3 mRNA and protein expression up-regulated in group C ( P ＜0.05＝. Conclusion COXs are involved in the up-regulation of the expression of P2X3 receptors in the DRG in rats with neuropathic pain, and the effect of COX-1 is stronger than that of COX-2. Key words: Prostaglandin-endoperoxide synthases; Neuralgia; Receptors, purinergic P2; Ganglia,spinal

Estimate of fibrosis in carbon tetrachloride induced chronic liver injury in rat by breath test with L‐[1‐13C] phenylalanine

Liver malfunction is a major problem in the world. Traditional diagnoses which are based on indices of blood biochemistry and liver biopsy have limitations and disadvantages. Developing new non‐invasive methods to accurately assess liver function is always an interesting topic. The objective of this study is to investigate the relationship between the parameters of breath test and the histological grading, and provide valid parameters for the evaluation of liver function. We injected L‐[1‐13C]phenylalaline (13C‐Phe) into rats in the control group (n=8) and carbon tetrachloride (CCl4 ) induced chronic injury group (n=21) through tail vein, quantified the 13CO2 using air isotope ratio mass spectrometry, and examined histological changes of rat livers. We tested 12 parameters and found that only unit‐liver‐weight (LW) parameters, including maximum unit abundance (13Cmax/LW), unit abundance at 2 min and 7 min (13C2/LW and 13C7/LW), cumulative unit 13C excretion in 10 min and 30 min (AUC10/LW and AUC30/LW) and unit 13C excretion rate constant (PheBT‐k/LW) were significantly affected in the chronic injury group. Our findings indicate that the breath test using 13C‐Phe (PheBT) together with the unit‐liver‐weight (LW) parameters can quantitatively reflect the degree of fibrosis in the livers of chronically injured rats.

Western blot quantification of aggrecan fragments in human synovial fluid indicates differences in fragment patterns between joint diseases

To develop a Western blot method for quantification of multiple aggrecan fragments in human synovial fluids (SFs). SF aggrecan fragments were prepared from knee healthy (reference), knee injury and arthritis subjects by CsCl gradient centrifugations collecting D1 fractions. Samples were analyzed by Western blot, using antibodies against the N-terminal epitope ARGS and the G3 domain, and fragments were quantified using a digital luminescence image analyzer. The method had a coefficients of variation of 10-30%, and a high correlation (r(S)=0.86) with a corresponding enzyme-linked immunosorbent assay (ELISA). The SFs from reference, knee injured and arthritic subjects contained two major ARGS fragments, ARGS-SELE and ARGS-CS1, and three major G3 fragments (GRGT-G3, GLGS-G3 and AGEG-G3). Compared to the reference, the acute arthritis and acute joint injury groups had a 30-fold elevated concentration of ARGS fragments, and both groups had a higher proportion of the aggrecan in joint fluid as ARGS fragments compared to the other groups. The reference and chronic injury groups had an excess of ARGS-CS1 fragments over ARGS-SELE fragments, while subjects with acute arthritis or osteoarthritis had a more even distribution between these fragments. We have developed a novel Western blot quantification method for quantification of SF aggrecan fragments which can differentiate fragments of different sizes sharing the same epitope. The anti-ARGS and anti-G3 quantitative Western blots provided information important for a better understanding of the proteolytic pathways in aggrecan breakdown, information that discriminates between different joint diseases, and may aid in identification of new biomarkers.

Attenuation of Kupffer Cell Function in Acute on Chronic Liver Injury Enhanced Engraftment of Transplanted Hepatocytes

The present study was designed to elucidate the relationship of engraftment efficiency of transplanted cells and Kupffer cell function in mice with acute on chronic liver injury and acute liver injury. The recipient dipeptidyl peptidase IV knockout (DPPIV(-/-)) mice were divided into two groups: (1) the acute on chronic liver injury group (CCl(4)/APAP group) that received CCl(4) (1 ml/kg) twice a week for 4 weeks following one dose of acetaminophen (APAP), 600 mg/kg; (2) the acute liver injury group (APAP-only group) that received a single dose of APAP at 600 mg/kg. DPPIV(+/+) hepatocytes were transplanted 24 h after APAP intoxication. Engraftment efficiency was evaluated at day 7 and day 14 after transplantation. The tumor necrosis factor-alpha (TNF-alpha) mRNA expression level of Kupffer cells immediately before cell transplantation was compared between the two groups before and after lipopolysaccharide (LPS, 100 ng/ml) stimulation. The number of transplanted cells and clusters in each 100x microscopic field were higher in the CCl(4)/APAP group at both day 7 (21.5 +/- 6.3 versus 8.3 +/- 4.0, p < 0.001; 14.9 +/- 4.6 versus 6.6 +/- 3.4, p < 0.001, respectively) and day 14 (17.3 +/- 4.4 versus 10.2 +/- 3.3, p = 0.001; 12.6 +/- 3.2 versus 7.9 +/- 1.6, p = 0.004, respectively). After LPS stimulation, the expression level of TNF-alpha was lower (175.7 +/- 54.6 versus 465.6 +/- 64.2, p = 0.002), and the increment of TNF-alpha expression was also less significant in the CCl(4)/APAP group (1.5-fold versus 6.5-fold, p = 0.014). Chronic liver injury desensitized Kupffer cells and reduced TNF-alpha expression, two results that correlated with the increased engraftment of transplanted cells.

Results of L‐[1‐13C]phenylalanine breath test with air isotope ratio mass spectrometry can reflect the activity of phenylalanine hydroxylase in cirrhotic rat liver

The L-[1-13C]phenylalanine breath test (PheBT) could potentially advance the evaluation of hepatocyte function and liver functional reserve. However, because the factors influencing PheBT results have not been clarified, the clinical application of the test has been limited. This study investigated the relationship between the parameters of PheBT, performed with air isotope ratio mass spectrometry, and the activity of phenylalanine hydroxylase (PAH), the phenylalanine metabolism rate-limiting enzyme, in rat liver, and proposes valid parameters for the assessment of liver function. Chronic injury to the liver was induced by the administration of CCl4 to male Sprague-Dawley rats for either 8 or 12 weeks. Livers from rats in the two cirrhotic groups were discolored, enlarged and roughly textured, with cells filled with fat granules of various sizes, pseudolobuli formations, and regenerated tubercles. Of the 12 parameters tested, only the unit liver weight (LW) breath test parameters, including the maximum abundance of 13C in breath (13Cmax/LW), 13C abundance in breaths 2 and 7 min after administration of L-[1-(13)C]phenylalanine (13C-phe) (13C2/LW and 13C7/LW), cumulative 13C excretion 10 and 30 min after 13C-phe administration (AUC10/LW and AUC30/LW), and the 13C excretion rate constant (PheBT-k/LW) were significantly affected in the chronic liver injury groups. There was no significant difference in the total PAH activity in liver among the three groups, but there was significant difference in unit LW PAH activity. Total PAH activity in the liver was significantly correlated with 13Cmax, 13C2, 13C7, AUC10, AUC30 and PheBT-k, while the unit LW PAH activity was significantly correlated with 13Cmax/LW, 13C2/LW, 13C7/LW, AUC10/LW, AUC30/LW and PheBT-k/LW. PheBT-k/LW was also correlated with biochemical indices that are used to assess liver function. The present findings indicate that the PheBT results based on air isotope ratio mass spectrometry can quantitatively reflect the change in total PAH activity in the livers of chronically injured rats. PheBT-k and PheBT-k/LW are the most sensitive among the test parameters, and can be used to assess liver functional reserve and hepatocyte damage at the molecular level.

This Month in Anesthesiology

This Month in Anesthesiology

Intravenous Glycine Inhibits the Micturition Reflex in Normal and Spinal Cord Injury Rats

We examined the influence of intravenous injection of glycine on the spinobulbospinal and spinal micturition reflexes in rats. Female rats were divided into an intact group and a chronic spinal cord injury (SCI) group with transection of the lower thoracic cord, and studied at 4 weeks after SCI. Under urethane anesthesia, isovolumetric cystometry was performed in each group before and after intravenous injection of glycine (0.01-100 mg/kg). Intravenous injection of glycine (0.1-100 mg/kg) transiently abolished bladder contractions after a delay of several minutes in both groups. In intact rats, intravenous injection of glycine prolonged the interval between bladder contractions, but did not change the amplitude of the contractions. On the other hand, intravenous glycine both prolonged the interval and decreased the amplitude of bladder contractions in SCI rats. However, intrathecal injection of low dose of strychnine (a selective glycine receptor antagonist; 0.001 pg) and following intravenous injection of glycine (0.1 mg/kg) did not affect the bladder contractions in intact rats. These results suggest that systemically administered glycine inhibits the afferent limb of the spinobulbospinal micturition reflex, and inhibits both the afferent and efferent limbs of the spinal micturition reflex at the lumbosacral cord level.

Articular cartilage lesions in ankles with lateral ligament injury. An arthroscopic study.

To investigate intraarticular lesions producing persistent postoperative pain, we arthroscopically examined 31 ankles in 31 patients (15 women and 16 men) with lateral ligament injury. The patients ranged in age from 15 to 33 years, with a mean of 20 years. Nine patients were freshly injured, and 22 patients had chronic injuries. All of the patients underwent arthroscopic examination immediately before the ligament operation. Chondral lesions were found in 89% of the freshly injured ankles and 95% of the ankles with chronic injuries. Most of these lesions were in the medial half of the ankle joint, especially in the anteromedial edge of the tibial plafond. After followup for 1 year postoperatively, persistent pain was noted in 4 patients who had chondral lesions of greater than one-half the thickness of the articular cartilage. Pain and tenderness were localized at the anteromedial joint line, corresponding to the location of the chondral lesions. Chondral lesions of greater than one-half the thickness of the articular cartilage were found in 8 ankles in the chronic injury group, but there were none in the fresh injury group. Thus, in lateral ligament injuries of the ankle, the longer the time elapsed from the initial injury, the more severe the associated chondral lesions became. These chondral lesions appear to cause persistent pain.