Acute gene inactivation in the adult mouse liver using the CRISPR-Cas9 technology.

Genetic manipulation in mice allows the discovery of gene function and biological mechanisms in vivo. The widely used Cre/LoxP system usually takes months to years especially when starting with the production of floxed alleles of a new gene of interest (GOI). Here, we describe a protocol using the CRISPR-Cas9 system to acutely inactivate the GOI in adult mice. This protocol enables hepatocyte-specific gene editing within 4 weeks in adult mice and avoids compensatory effects of traditional gene inactivation initiated during various developmental stages. For complete details on the use and execution of this protocol, please refer to Wang et al. (2020).

Stereological study on the numerical plasticity of myelinated fibers and oligodendrocytes in the rat spinal cord with painful diabetic neuropathy.

Painful diabetic neuropathy may associate with nerve morphological plasticity in both peripheral and central nervous system. The aim of this study was to determine numerical changes of myelinated fibers in the spinothalamic tract region and oligodendrocytes in the spinal dorsal horn of rats with painful diabetic neuropathy and the effects of metformin on the above changes. Male Sprague-Dawley rats were randomly allocated into the control group (n = 7), the painful diabetic neuropathy group (n = 6) and the painful diabetic neuropathy treated with metformin group (the PDN + M group, n = 7), respectively. Twenty-eight days after medication, numbers of myelinated fibers in the spinothalamic tract and oligodendrocytes in the spinal dorsal horn were estimated by the optical disector (a stereological technique). Compared to the control group, number of myelinated fibers in the spinothalamic tract increased significantly in the painful diabetic neuropathy and PDN + M group, compared to the painful diabetic neuropathy group, number of myelinated fibers decreased in the PDN + M group (P < 0.05). As the oligodendrocyte in the spinal dorsal horn was considered, its number increased significantly in the painful diabetic neuropathy group compared to the control and the PDN + M group (P < 0.05), there was no significant difference between the control and the PDN + M group (P > 0.05). Our results indicate that painful diabetic neuropathy is associated with a serial of morphometric plasticity in the rat spinal cord including the numerical increase of the myelinated fibers in the spinothalamic tract and the oligodendrocytes in the spinal dorsal horn. The analgesic effect of metformin against painful diabetic neuropathy might be related to its adverse effects on the above morphometric plasticity.

Comparative study of serum levels of BMP-2 and heterotopic ossification in traumatic brain injury and fractures patients / 中国骨伤

<p><b>OBJECTIVE</b>To investigate the relationship between serum level of bone morphogenetic proteins-2 (BMP-2) and heterotopic ossification (HO) in traumatic brain injury (TBI) and fractures patients, providing the theoretical evidence for the clinical prevention of HO.</p><p><b>METHODS</b>From December 2007 to January 2009, 145 with trama patients were selected. There were 96 closed primary traumatic brain injury patients, 1 penetrating primary traumatic brain injury, 29 fractures of the radius and ulna, 11 fractures of the humerus, 32 fractures of the tibia and fibula, 27 fractures of the femur. All patients were divided into three groups (i.e., group A, group B and group C) by the type of fracture. Fifty-seven patients in group A (TBI only), including 37 males and 20 females, ranged in the age from 29 to 61 years, with an average age of (43.91 +/- 11.09) years. The disease course was from 13 to 67 d, with an average duration of (18.96 +/- 10.46) d. Forty-eight patients in group B (fractures only), 25 males and 23 females, ranged in age from 31 to 54 years, with an average age of (41.73 +/- 8.41) years. The disease course was from 6 to 48 d, with an average duration of (16.02 +/- 8.71) d. Forty patients in group C (TBI combined with extremities fractures), including 23 males and 17 females, ranged in age from 30 to 60 years, with an average age of (45.87 +/- 14.15) years. The disease course was from 18 to 76 d, with an average of (21.28 +/- 13.02) d. Thirty-one extremities fractures with no significant separations or displacements of fragments were treated with traction reductions, cast immobilization or splint fixations. Sixty-eight fractures with significant separations and displacements of fragments were treated with intramedullary nail fixations or screw internal fixations. Sixty-three TBI patients were treated with open-skull surgeries immediately while 34 TBI patients were treated with stanching bleeding, reducing intracranial pressure and improving cerebral blood circulation. All patients were also divided into two groups (group D and group E) according to the 14-to 16-month follow-up X-ray film results. Seventeen patients in group D (HO had been found), including 11 males and 6 females, ranged in age from 29 to 55 years old, with an average age of (46.88 +/- 7.13) years. The disease course was from 6 to 30 d, with an average of (20.18 +/- 9.78) d. All 128 patients in group E (HO had been not found), including 74 males and 54 females, ranged in age from 33 to 61 years, with an average age of (43.31 +/- 12.94) years. The disease course was from 15 to 76 d, with an average of (18.42 +/- 11.58) d. The 49 subjects in group F (normal controls), 29 males and 20 females, ranged in age from 31 to 60 years, average (43.50 +/- 14.40) years. Peripheral blood samples were taken for the determination of BMP-2 in blood serum on 0.5, 3, 15 d and 30 d after fractures by enzyme-linked immunosorbent assay (ELISA). Analysis of variance and least significant difference test were done with the help of SPSS 13.0 statistic software.</p><p><b>RESULTS</b>The incidence rates of HO between the TBI only patients (21.05%, 12/57) and the fractures only patients(4.17%, 2/48) were significant different (chi2=5.05, P<0.05). Serum levels of BMP-2 at 0.5, 3 d and 15 d between group A and group B were significant different. Serum levels of BMP-2 at 0.5, 3, 15 d and 30 d between group D and group E were significant different. Serum levels of BMP-2 at each time in each group were higher than the control group (51.30 +/- 23.41 ng/L) (P<0.01).</p><p><b>CONCLUSION</b>High serum levels of BMP-2 in TBI only group is one of factors in causing HO. Serum level of BMP-2 at 15 d since fractures may be the obvervational index of HO prevention.</p>