[Influence of electroacupuncture on the expression of AMPA receptor subunit GluR1 in the spinal injured area of the rats with acute spinal cord injury].

OBJECTIVE: To explore the influence of electroacupuncture (EA) on the expression of AMPA receptor subunit GluR1 in the rats with acute spinal cord injury (SCI) and explore the potential effect mechanism of EA in treatment of acute SCI. METHODS: A total of 80 SD rats were randomly divided into five groups, i.e. a sham-operation group, a model group, an AMPA antagonist (DNQX) group, an EA group and a DNQX+EA group, 16 rats in each group. The modified Allen's impacting method was adopted to prepare the rat model of acute SCI at T10. In the DNQX group, the intrathecal injection of 10 µL DNQX solution with a concentration of 1 nmol/µL was administered in 0.5 h after modeling success. In the EA group, EA (disperse-dense wave, 2 Hz/100 Hz in frequency, 0.5 mA in output current) was given at "Dazhui" (GV 14) and "Mingmen" (GV 4) in 0.5 h, 12 h and 24 h after modeling success for 30 min and totally 3 times. In the DNQX + EA group, the interventions in the above two groups were managed. The Basso, Beattie and Bresnahan locomotor rating score (BBB) was applied to evaluate the changes of locomotor function in the rats before modeling and in 6 h, 24 h and 48 h after modeling successively. Using the hematoxylin-eosin (HE) staining, the histopathological changes in the spinal anterior horn were observed in the spinal injured area. The immunofluorescence method was adopted to determine the number of GluR1 positive neuron of the spinal anterior horn. The Western blot method was used to determine the protein expression of GluR1 in the injured area. RESULTS: Compared to the sham-operation group in 6 h, 24 h and 48 h after modeling, the BBB scores were all significantly decreased in the model group (P<0.001) at the corresponding points. The BBB score was increased in each of intervention groups, but without statistical difference as compared with the model group (P>0.05). In the model group, it was found that the boundary between gray matter and white matter in the spinal anterior horn was blurred, the interstitial space enlarged, the neuron volume obviously shrunken, the cytoplasm decreased, the red stain deepened and some neuron nuclei fixed and shrunk. In the EA group, the morphology of the spinal anterior horn in the injured area was improved obviously, which was similar in the DNQX group and the DNQX + EA group. Compared with the sham-operation group, the GluR1 protein expression in the spinal injury area was increased (P<0.001) and the number of GluR1 positive neurons elevated (P<0.001) in the spinal anterior horn in the model group. Compared with the model group, in the EA group, the DNQX group and the DNQX + EA group, GluR1 protein expression was decreased (P<0.05, P<0.01) and the number of GluR1 positive neurons in the spinal anterior horn reduced (P<0.001). CONCLUSION: The intervention with EA at "Dazhui" and "Mingmen" promotes the repair of the injured nerve in the spinal anterior horn probably through inhibiting GluR1 expression in the spinal injured area in the rats with acute SCI.

Influence of electroacupuncture on the expression of AMPA receptor subunit GluR1 in the spinal injured area of the rats with acute spinal cord injury / 中国针灸

OBJECTIVE@#To explore the influence of electroacupuncture (EA) on the expression of AMPA receptor subunit GluR1 in the rats with acute spinal cord injury (SCI) and explore the potential effect mechanism of EA in treatment of acute SCI.@*METHODS@#A total of 80 SD rats were randomly divided into five groups, i.e. a sham-operation group, a model group, an AMPA antagonist (DNQX) group, an EA group and a DNQX+EA group, 16 rats in each group. The modified Allen's impacting method was adopted to prepare the rat model of acute SCI at T@*RESULTS@#Compared to the sham-operation group in 6 h, 24 h and 48 h after modeling, the BBB scores were all significantly decreased in the model group (@*CONCLUSION@#The intervention with EA at "Dazhui" and "Mingmen" promotes the repair of the injured nerve in the spinal anterior horn probably through inhibiting GluR1 expression in the spinal injured area in the rats with acute SCI.

[Influence of electroacupuncture of "Dazhui" (EX-B2) and "Mingmen" (GV4) on NR2B expression in anterior horns of spinal cord in rats with acute spinal cord injury].

OBJECTIVE: To observe the influence of eletroacupuncture (EA) at "Dazhui" (EX-B2) and "Mingmen" (GV4) on expression of NR2B subunit of N-methyl-D-aspartate receptor (NMDA) in the injured anterior horn (AH) area of rats with acute spinal cord injury (SCI), so as to explore its mechanisms underlying improvement of neural repair. METHODS: A total of 96 male Sprague-Dawley (SD) rats were randomly and equally divided into four groups: sham operation (sham), model, medication (Methyl-prednisone, MP) and EA (n＝24 in each group). The acute SCI model was established by using a MASCIS spinal cord impactor. EA (2 Hz, 0.5 mA) was applied to EX-B2 and GV4 for 30 min, once at 0.5 h, 12 and 24 h after SCI. Rats of the medication group were treated by tail intravenous injection of MP 30 mg/kg within 15 min (impact therapy) and 5.4 mg•kg－1•h－1 (maintaining treatment) 45 min thereafter for 23 h. The Basso, Beattie and Bresnahan (BBB) rating scale (0 to 21 points) was used to assess changes of locomotor function 6, 24 and 48 h after SCI. Histopathological changes of the injured spinal cord AH region were observed after sectioning and hematoxylin-eosin (H.E.) staining, and the expression levels of NR2B mRNA and protein of AH were measured by quantitative real-time PCR, Western blot and immunofluorescence, respectively. RESULTS: After SCI, the BBB scores at 6, 24 and 48 h were significantly decreased in the model group compared with those of the sham group (P<0.001), but had no remarkable changes in both medication and EA groups after the intervention in comparison with those of the model group (P>0.05). After modeling, the histopathological changes (blurred border of the grey-white matter, cellular karyopyknosis, deepening of the cytoplasmic red stain, and rupture, dissolution and disordered arrangement of myelinated nerve fibers) in the injury area of the spinal cord in the model group were apparent, the number of NR2B positive neurons and the relative expression levels of NR2B mRNA and protein were significantly increased in the model group relevant to the sham group (P<0.05). In contrast to the model group, the injured severity of the spinal cord AH region was relatively milder, and the expression levels of NR2B mRNA and protein were considerably down-regulated in both EA and MP groups (P<0.05). However, there were no significant differences between the EA and MP groups in the expression levels of NR2B mRNA and protein (P>0.05). CONCLUSION: EA at EX-B2 and GV4 may inhibit the expression of NR2B mRNA and protein in acute SCI rats, which may contribute to its action in promoting nerve repair of the injured ventricolumna area of the thoracic spinal cord.

Influence of electroacupuncture of "Dazhui" (EX-B2) and "Mingmen" (GV4) on NR2B expression in anterior horns of spinal cord in rats with acute spinal cord injury / 针刺研究

OBJECTIVE: To observe the influence of eletroacupuncture (EA) at "Dazhui" (EX-B2) and "Mingmen" (GV4) on expression of NR2B subunit of N-methyl-D-aspartate receptor (NMDA) in the injured anterior horn (AH) area of rats with acute spinal cord injury (SCI), so as to explore its mechanisms underlying improvement of neural repair. METHODS: A total of 96 male Sprague-Dawley (SD) rats were randomly and equally divided into four groups: sham operation (sham), model, medication (Methyl-prednisone, MP) and EA (n＝24 in each group). The acute SCI model was established by using a MASCIS spinal cord impactor. EA (2 Hz, 0.5 mA) was applied to EX-B2 and GV4 for 30 min, once at 0.5 h, 12 and 24 h after SCI. Rats of the medication group were treated by tail intravenous injection of MP 30 mg/kg within 15 min (impact therapy) and 5.4 mg•kg－1•h－1 (maintaining treatment) 45 min thereafter for 23 h. The Basso, Beattie and Bresnahan (BBB) rating scale (0 to 21 points) was used to assess changes of locomotor function 6, 24 and 48 h after SCI. Histopathological changes of the injured spinal cord AH region were observed after sectioning and hematoxylin-eosin (H.E.) staining, and the expression levels of NR2B mRNA and protein of AH were measured by quantitative real-time PCR, Western blot and immunofluorescence, respectively. RESULTS: After SCI, the BBB scores at 6, 24 and 48 h were significantly decreased in the model group compared with those of the sham group (P0.05). After modeling, the histopathological changes (blurred border of the grey-white matter, cellular karyopyknosis, deepening of the cytoplasmic red stain, and rupture, dissolution and disordered arrangement of myelinated nerve fibers) in the injury area of the spinal cord in the model group were apparent, the number of NR2B positive neurons and the relative expression levels of NR2B mRNA and protein were significantly increased in the model group relevant to the sham group (P0.05). CONCLUSION: EA at EX-B2 and GV4 may inhibit the expression of NR2B mRNA and protein in acute SCI rats, which may contribute to its action in promoting nerve repair of the injured ventricolumna area of the thoracic spinal cord.

Dynamic changes of myeloid-derived suppressor cells and regulatory T cells in livers of mice infected with Echinococcus granulosus / 中国血吸虫病防治杂志

Objective To investigate the dynamics changes of the myeloid-derived suppressor cells (MDSCs) and regulatory T (Treg) cells in mice infected with Echinococcus granulosus and explore the possible biological significance. Methods Thirty female BALB/c mice of 6 weeks old were randomly divided into the infection and control groups, of 15 mice in each group. Mice in the infection group were intraperitoneally injected with 2 000 E. granulosus protoscoleces, while those in the control group were injected with the same volume of physiological saline. Mouse liver white blood cells were harvested 3 (early stage), 6 (medium stage) and 12 months (late stage) post-infection, and the proportions of MDSCs, their subpopulations (M-MDSCs and PMN-MDSCs) and Treg cells were assessed by flow cytometry. Results The proportions of MDSCs were (1.61 ± 0.36)%, (5.68 ± 0.69)% and (16.18 ± 0.69)% in mouse liver white blood cells in the infection group 3, 6 and 12 months post-infection with E. granulosus, and (2.19 ± 0.42)%, (0.99 ± 0.07) % and (4.18 ± 0.84)% in the control group, and there were significant differences in the proportion of the MDSCs in mouse liver white blood cells between the infection and control groups 6 and 12 months post-infection (P < 0.01). The proportions of M-MDSCs were (0.69 ± 0.27)%, (5.30 ± 0.72)% and (10.75 ± 0.29)% in mouse liver white blood cells in the infection group 3, 6 and 12 months post-infection, and (0.42 ± 0.24)%, (0.69 ± 0.02)% and (2.12 ± 0.13)% in the control group, and there were significant differences in the proportion of the M-MDSCs in the mouse liver white blood cells between the infection and control groups 6 and 12 months post-infection (P < 0.01). The proportions of PMN-MDSCs were (0.93 ± 0.23)%, (0.32 ± 0.02)% and (5.14 ± 1.03)% in mouse liver white blood cells in the infection group 3, 6 and 12 months post-infection, and (1.77 ± 0.26)%, (0.28 ± 0.05)% and (1.99 ± 0.90)% in the control group, and there were significant differences in the proportion of PMN-MDSCs in mouse liver white blood cells between the infection and control groups 3 and 12 months post-infection (P < 0.05). The proportions of Treg cells were (3.35 ± 0.14)%, (6.24 ± 0.38)% and (3.41 ± 0.07)% in mouse liver white blood cells in the infection group 3, 6 and 12 months post-infection, and (3.48 ± 0.46)%, (3.65 ± 0.45)% and (3.12 ± 0.12)% in the control group, and there were significant differences in the proportion of Treg cells in mouse liver white blood cells between the infection and control groups 6 and 12 months post-infection (P < 0.01). Conclusions The percentages of both MDSCs and Treg cells increase in mouse liver white blood cells 6 and 12 months post-infection with E. granulosus, and a more remarkable increase is seen in the percentage of MDSCs, which is mainly found in M-MDSCs. These findings suggest that M-MDSCs may play a major immunosuppressive role in the medium and late stages of E. granulosus infection in mice.

Dynamic changes of myeloid-derived suppressor cells and regulatory T cells in livers of mice infected with Echinococcus granulosus / 中国血吸虫病防治杂志

Objective To investigate the dynamics changes of the myeloid-derived suppressor cells (MDSCs) and regulatory T (Treg) cells in mice infected with Echinococcus granulosus and explore the possible biological significance. Methods Thirty female BALB/c mice of 6 weeks old were randomly divided into the infection and control groups, of 15 mice in each group. Mice in the infection group were intraperitoneally injected with 2 000 E. granulosus protoscoleces, while those in the control group were injected with the same volume of physiological saline. Mouse liver white blood cells were harvested 3 (early stage), 6 (medium stage) and 12 months (late stage) post-infection, and the proportions of MDSCs, their subpopulations (M-MDSCs and PMN-MDSCs) and Treg cells were assessed by flow cytometry. Results The proportions of MDSCs were (1.61 ± 0.36)%, (5.68 ± 0.69)% and (16.18 ± 0.69)% in mouse liver white blood cells in the infection group 3, 6 and 12 months post-infection with E. granulosus, and (2.19 ± 0.42)%, (0.99 ± 0.07) % and (4.18 ± 0.84)% in the control group, and there were significant differences in the proportion of the MDSCs in mouse liver white blood cells between the infection and control groups 6 and 12 months post-infection (P < 0.01). The proportions of M-MDSCs were (0.69 ± 0.27)%, (5.30 ± 0.72)% and (10.75 ± 0.29)% in mouse liver white blood cells in the infection group 3, 6 and 12 months post-infection, and (0.42 ± 0.24)%, (0.69 ± 0.02)% and (2.12 ± 0.13)% in the control group, and there were significant differences in the proportion of the M-MDSCs in the mouse liver white blood cells between the infection and control groups 6 and 12 months post-infection (P < 0.01). The proportions of PMN-MDSCs were (0.93 ± 0.23)%, (0.32 ± 0.02)% and (5.14 ± 1.03)% in mouse liver white blood cells in the infection group 3, 6 and 12 months post-infection, and (1.77 ± 0.26)%, (0.28 ± 0.05)% and (1.99 ± 0.90)% in the control group, and there were significant differences in the proportion of PMN-MDSCs in mouse liver white blood cells between the infection and control groups 3 and 12 months post-infection (P < 0.05). The proportions of Treg cells were (3.35 ± 0.14)%, (6.24 ± 0.38)% and (3.41 ± 0.07)% in mouse liver white blood cells in the infection group 3, 6 and 12 months post-infection, and (3.48 ± 0.46)%, (3.65 ± 0.45)% and (3.12 ± 0.12)% in the control group, and there were significant differences in the proportion of Treg cells in mouse liver white blood cells between the infection and control groups 6 and 12 months post-infection (P < 0.01). Conclusions The percentages of both MDSCs and Treg cells increase in mouse liver white blood cells 6 and 12 months post-infection with E. granulosus, and a more remarkable increase is seen in the percentage of MDSCs, which is mainly found in M-MDSCs. These findings suggest that M-MDSCs may play a major immunosuppressive role in the medium and late stages of E. granulosus infection in mice.

KCC2-GABAA pathway correlates with the analgesic effect of electro-acupuncture in CCI rats.

Potassium-chloride cotransporter 2 (KCC2) has been indicated to serve a crucial role during chronic neuropathic pain (NP). Following the emergence of NP, γ­aminobutyric acid (GABA) A receptor­mediated signaling may be further impaired by the changes of KCC2 chloride anion gradient. In the present study, the authors investigate the effect of electro-acupuncture (EA) on the behavior and the expression of KCC2 and GABAA receptor γ2 subunit in the spinal cord of chronic constriction injury (CCI) model rats. A total of 60 adult male Sprague­Dawley rats were divided into four groups: Normal group, sham­CCI group, CCI group and CCI+EA group. The effect of EA was assessed via the values of mechanical withdrawal threshold and thermal withdrawal latency, which were significantly improved upon stimulation of the ST­36 and GB­34 acupoints. In addition, a marked reduction in both the mRNA and protein levels of KCC2 and GABAA receptor γ2 subunit was observed in the spinal cord following loose ligation of the sciatic nerve. The reductions in KCC2 and GABAA receptor γ2 subunit expression were reversed by EA treatment. These results support the notion that KCC2 and GABAA receptor γ2 subunit contribute to NP following peripheral nerve injury and extend the understanding of the analgesic effects of EA on NP.

The regulatory effect of electro-acupuncture on the expression of NMDA receptors in a SCI rat model.

BACKGROUND: In early spinal cord injury (SCI), glutamate receptors, including N-methyl-d-aspartate (NMDA) receptors (NMDARs), are over-stimulated by excessively released glutamate. The enhanced activity of NMDARs may cause cell death by overloading calcium (Ca2+) into cells based on their high permeability to Ca2+. Studies in SCI animals have shown that treatment with electro-acupuncture (EA) is able to reduce cell death and to improve functional recovery. One possible mechanism of this neuroprotective effect is that EA has regulatory effect on NMDARs. AIMS: To test whether EA could protect the spinal cord after SCI by decreasing the expression levels of NR1 and NR2A. MAIN METHODS: We conducted EA treatment on a rat SCI model produced with a New York University (NYU) Impactor and measured hindlimb locomotor function by Basso, Beattie and Bresnahan Locomotor Rating Scale (BBB Scale). The expression of NR1 and NR2, the subunits of NMDARs, in the injured spinal cord was measured by Immunofluorescence stainings, western blot and real-time quantitative PCR (RT-qPCR). KEY FINDING: Our results showed that two days after the SCI the expression of NR1 and NR2 were dramatically enhanced at both protein and mNRA levels, which were significantly reduced by EA treatment at two specific acupoints, Dazhui (DU14) and Mingmen (DU4). SIGNIFICANCE: EA is a potential therapeutic method for treating early SCI in human.

Interleukin-4- and NACHT, LRR and PYD domains-containing protein 3-independent mechanisms of alum enhanced T helper type 2 responses on basophils.

Aluminium hydroxide (alum), the most widely used adjuvant in human and animal vaccines, has long been known to promote T helper type 2 (Th2) responses and Th2-associated humoral responses, but the mechanisms have remained poorly understood. In this study, we explored whether alum is able to directly modulate antigen-presenting cells to enhance their potency for Th2 polarization. We found that alum treatment of dendritic cells failed to show any Th2-promoting activities. In contrast, alum was able to enhance the capacity of basophils to induce Th2 cells. When basophils from interleukin-4 (IL-4) knockout mice were examined, the intrinsic Th2-promoting activities by basophils were largely abrogated, but the alum-enhanced Th2-promoting activities on basophils were still detectable. More importantly, Th2-promoting adjuvant activities by alum found in IL-4 knockout mice were also largely reduced when basophils were depleted by antibody administration. Therefore, basophils can mediate Th2-promoting activities by alum both in vitro and in vivo through IL-4-independent mechanisms. Further studies revealed that secreted soluble molecules from alum-treated basophils were able to confer the Th2-promoting activities, and neutralization of thymic stromal lymphopoietin or IL-25 attenuated the IL-4-independent development of Th2 cells elicited by alum-treated basophils. Finally, alum was able to activate NACHT, LRR and PYD domains-containing protein 3 (NLRP3) inflammasome in murine basophils in the same way as alum in professional antigen-presenting cells, but NLRP3 was not required for Th2-promoting activities on basophils by alum in vitro. These results demonstrated that alum can enhance the capacities of basophils to polarize Th2 cells via IL-4- and NLRP3-independent pathways.

IL-4-Producing Dendritic Cells Induced during Schistosoma japonica Infection Promote Th2 Cells via IL-4-Dependent Pathway.

Although dendritic cells (DCs) have been widely demonstrated to play essential roles in initiation of Th2 responses in helminth infections and allergic reactions, the mechanisms remain uncertain largely because DCs do not produce IL-4. In present investigation, we have uncovered a novel subset of DCs from mice infected with Th2-provoking pathogens Schistosoma japonica, which independently promoted Th2 cells via IL-4-dependent pathway. These DCs contained similar levels of IL-4 mRNA and higher levels of IL-12p40 mRNA comparing to basophils, correlating to their Th2-promoting and Th1-promoting dual polarization capacities. Characterized by expression of FcεRI(+), these DCs were induced independent of T cells. Further investigations revealed that Th2-promoting FcεRI(+) DCs were monocyte-derived inflammatory DCs, which were sufficient to induce Th2 cells in vivo. Egg Ags together with GM-CSF or IL-3 alone were able to stimulate the generation of Th2-promoting FcεRI(+) DCs from bone marrow cells in vitro. To our knowledge, our data for the first time demonstrate that IL-4-producing DCs are induced under some Th2-provoking situations, and they should play important roles in initiation of Th2 response.

Cu-Cu2O-TiO2 nanojunction systems with an unusual electron-hole transportation pathway and enhanced photocatalytic properties.

Multicomponent Cu-Cu2O-TiO2 nanojunction systems were successfully synthesized by a mild chemical process, and their structure and composition were thoroughly analyzed by X-ray diffraction, transmission electron microscopy, field-emission scanning electron microscopy, and X-ray photoelectron spectroscopy. The as-prepared Cu-Cu2O-TiO2 (3 and 9 h) nanojunctions demonstrated higher photocatalytic activities under UV/Vis light irradiation in the process of the degradation of organic compounds than those of the Cu-Cu2O, Cu-TiO2, and Cu2O-TiO2 starting materials. Moreover, time-resolved photoluminescence spectra demonstrated that the quenching times of electrons and holes in Cu-Cu2O-TiO2 (3 h) is higher than that of Cu-Cu2O-TiO2 (9 h); this leads to a better photocatalytic performance of Cu-Cu2O-TiO2 (3 h). The improvement in photodegradation activity and electron-hole separation of Cu-Cu2O-TiO2 (3 h) can be ascribed to the rational coupling of components and dimensional control. Meanwhile, an unusual electron-hole transmission pathway for photocatalytic reactions over Cu-Cu2O-TiO2 nanojunctions was also identified.

From titanium oxydifluoride (TiOF2) to titania (TiO2): phase transition and non-metal doping with enhanced photocatalytic hydrogen (H2) evolution properties.

Single-crystalline TiOF(2) crystals with cubical morphology were prepared via a facile solvothermal method and their transformation to anatase TiO(2) under different calcination conditions such as pure argon, moist argon and pure hydrogen sulfide (H(2)S) was explored by using XRD/Raman/UV-Vis/SEM/TEM/SAED. The non-metal sulfur doping was successfully fulfilled and the doped TiO(2) microcubes showed the best photocatalytic H(2) evolution property.

Synthesis of micro-sized titanium dioxide nanosheets wholly exposed with high-energy {001} and {100} facets.

A new synthetic strategy was developed to prepare large-sized well-defined anatase TiO(2) nanosheets wholly dominated with thermodynamically unfavorable high-reactive {001} and {100} facets, which has a percentage of 98.7% and 1.3%, respectively. The as-prepared anatase TiO(2) nanosheets show a well-faceted morphology and have a large size in length (ca. 4.14 µm). The formation mechanism of the anatase TiO(2) nanosheets was also analyzed and investigated.

[Seasonal variations of HCOOH and HCHO in precipitation in Guiyang].

Formic acid and formaldehyde are the important and ubiquitous chemical constituents in the atmosphere. Precipitation in Guiyang, the capital city of Guizhou province, was analyzed with ICS-90 ion chromatography for their concentration changes in a year. The volume mean weight average concentration (VMWA) is (13.27 +/- 25.92) micromol/L and (7.89 +/- 8.92) micromol/L for formic acid and formaldehyde, respectively. Both species demonstrated higher concentration during winter-half-year than the summer-half-year. Day-time concentration is higher than the night-time, the diurnal variations for both chemical constituents indicate the sources from human activities and photochemical reactions. Regression analysis of concentration vs precipitation volume and deposition amount vs precipitation volume reveals that dilution and scavenging of precipitation in the below-cloud process are responsible for the changes of the concentrations. The significant correlation between formic acid and formaldehyde suggests similar sources or reaction relationship. The known aqueous oxidation of formaldehyde, however, can not be used to explain the link of the two compounds, and thus should not be the major source of formic acid in Guiyang, Instead, photochemical reactions of unsaturated hydrocarbons (natural or anthropological) were the major sources for formic acid and formaldehyde.

Molecular evidence for species-level distinctions in clouded leopards.

Among the 37 living species of Felidae, the clouded leopard (Neofelis nebulosa) is generally classified as a monotypic genus basal to the Panthera lineage of great cats. This secretive, mid-sized (16-23 kg) carnivore, now severely endangered, is traditionally subdivided into four southeast Asian subspecies (Figure 1A). We used molecular genetic methods to re-evaluate subspecies partitions and to quantify patterns of population genetic variation among 109 clouded leopards of known geographic origin (Figure 1A, Tables S1 ans S2 in the Supplemental Data available online). We found strong phylogeographic monophyly and large genetic distances between N. n. nebulosa (mainland) and N. n. diardi (Borneo; n = 3 individuals) with mtDNA (771 bp), nuclear DNA (3100 bp), and 51 microsatellite loci. Thirty-six fixed mitochondrial and nuclear nucleotide differences and 20 microsatellite loci with nonoverlapping allele-size ranges distinguished N. n. nebulosa from N. n. diardi. Along with fixed subspecies-specific chromosomal differences, this degree of differentiation is equivalent to, or greater than, comparable measures among five recognized Panthera species (lion, tiger, leopard, jaguar, and snow leopard). These distinctions increase the urgency of clouded leopard conservation efforts, and if affirmed by morphological analysis and wider sampling of N. n. diardi in Borneo and Sumatra, would support reclassification of N. n. diardi as a new species (Neofelis diardi).