Advances in the application of mesenchymal stem cells, exosomes, biomimetic materials, and 3D printing in osteoporosis treatment.

Owing to an increase in the aging population, osteoporosis has become a severe public health concern, with a high prevalence among the elderly and postmenopausal adults. Osteoporosis-related fracture is a major cause of morbidity and mortality in elderly and postmenopausal adults, posing a considerable socioeconomic burden. However, existing treatments can only slow down the process of osteoporosis, reduce the risk of fractures, and repair fractures locally. Therefore, emerging methods for treating osteoporosis, such as mesenchymal stem cell transplantation, exosome-driving drug delivery systems, biomimetic materials, and 3D printing technology, have received increasing research attention, with significant progress. Mesenchymal stem cells (MSCs) are pluripotent stem cells that can differentiate into different types of functional cells. Exosomes play a key role in regulating cell microenvironments through paracrine mechanisms. Bionic materials and 3D printed scaffolds are beneficial for the reconstruction and repair of osteoporotic bones and osteoporosis-related fractures. Stem cells, exosomes, and biomimetic materials represent emerging technologies for osteoporosis treatment. This review summarizes the latest developments in these three aspects.

Repression of let-7b-5p prevents the development of multifidus muscle dysfunction by promoting vitamin D accumulation via upregulation of electron transfer flavoprotein alpha subunit in a rat model of multifidus muscle injury.

Multifidus muscle dysfunction is associated with the multifidus muscle injury (MMI), which ultimately result in the low-back pain. Increasing evidence shows that microRNAs (miRs) may be involved in multifidus muscle dysfunction. In this study, we tested the hypothesis that downregulation of let-7b-5p may inhibit the multifidus muscle dysfunction development and progression. The target prediction program and luciferase activity determination confirmed electron transfer flavoprotein alpha subunit (ETFA) as a direct target gene of let-7b-5p. To study the mechanisms and functions of let-7b-5p in relation to ETFA in MMI progression, we prepared rats with experimental MMI, and a lentivirus-based packaging system was designed to upregulate expressions of let-7b-5p, and downregulate the expression of ETFA. ETFA was identified as a target gene of let-7b-5p. Older age, a longer duration of pain, and higher visual analog scale and Oswestry disability index scores for the patients with chronic low-back pain were linked to a more severe degree of degenerative muscle atrophy and fatty infiltration. Increased expression of let-7b-5p and decreased expression of ETFA and vitamin D receptor (VDR) were positively correlated with multifidus muscle dysfunction. Downregulated let-7b-5p could inhibit infiltration of collagen fibers, reverse the ultrastructural changes of multifidus muscle, and induce the VDR expression, thereby repair the MMI. The results provided a potential basis for let-7b-5p that could support targeted intervention in multifidus muscle dysfunction. Collectively, this study confirmed that downregulation of let-7b-5p has a potential inhibitory effect on the development of the function of the musculus myocytes by upregulating ETFA.

LncRNA DLX6-AS1/miR-129-5p/DLK1 axis aggravates stemness of osteosarcoma through Wnt signaling.

Osteosarcoma is the most common primary bone tumor and occurs most frequently in adolescents. Cancer stem cells (CSCs) are resistant to chemotherapy and radiotherapy, and can drive cancer recurrence. In this study, we aimed to investigate the effect of Long noncoding RNAs (lncRNAs) DLX6-AS1 on osteosarcoma stemness and the underlying mechanism involved. DLX6-AS1 enhanced osteosarcoma stemness in vitro and in vivo. Moreover, DLX6-AS1 competitively interacted with miR-129-5p to DLK1, resulting in activation of Wnt signaling and promotion of stemness in osteosarcoma. DLX6-AS1 functionally interplayed with miR-129-5p to form a reciprocal feedback loop to activate Wnt signaling. High DLX6-AS1 expression was observed in osteosarcoma tissues, and predicted a poor prognosis for osteosarcoma patients. Our study suggests that DLX6-AS1, combined with miR-129-5p and DLK1, can be utilized as factors for the clinical diagnosis and prognosis of osteosarcoma, and may be potential targets for the treatment of osteosarcoma.

Spinal disease in myeloma: cohort analysis at a specialist spinal surgery centre indicates benefit of early surgical augmentation or bracing.

BACKGROUND: Multiple myeloma osteolytic disease affecting the spine results in vertebral compression fractures. These are painful, result in kyphosis, and impact respiratory function and quality of life. We explore the impact of time to presentation on the efficacy of spinal treatment modalities. METHODS: We retrospectively reviewed 183 patients with spinal myeloma presenting to our service over a 2 year period. RESULTS: Median time from multiple myeloma diagnosis to presentation at our centre was 195 days. Eighty-four patients (45.9 %) were treated with balloon kyphoplasty and the remainder with a thoracolumbar-sacral orthosis as per our published protocol. Patients presenting earlier than 195 days from diagnosis had significant improvements in patient reported outcome measures: EuroQol 5-Dimensions (p < 0.001), Oswestry Disability Index (p < 0.001), and Visual Analogue Pain Score (p < 0.001) at follow-up, regardless of treatment. Patients presenting after 195 days, however, only experienced benefit following balloon kyphoplasty, with no significant benefit from non-operative management. CONCLUSION: Vertebral augmentation and thoracolumbar bracing improve patient reported outcome scores in patients with spinal myeloma. However, delay in treatment negatively impacts clinical outcome, particularly if managed non-operatively. It is important to screen and treat patients with MM and back pain early to prevent deformity and improve quality of life.

TGF-ß1 precursor and CD8 are potential prognostic and predictive markers in operated breast cancer.

The transforming growth factor ß1 (TGF-ß1) and CD8-positive T cells are two important immune factors that function at opposite directions. The purpose of this study was to verify the relationship between the two factors and their associations with long-term effects of adjuvant chemotherapy or endocrine therapy in breast cancer. Expression of TGF-ß1 precursor and CD8 was immunohistochemically detected on surgically-obtained tumor samples of 130 (stage I-III) invasive breast carcinomas from Chinese subjects, who were followed up for a mean time of 112 months. Interstitial CD8-positive cells and TGF-ß1 precursor-positive cells adjacent to tumor nests were counted. Infiltration of CD8-positive lymphocytes into tumor nests and TGF-ß1 precursor expression in tumor cells were observed and survival analysis was performed. Our results showed that density of interstitial CD8-positive lymphocytes was an independent adverse prognostic factor for distant disease-free survival (DDFS) (HR=8.416, 95% CI=1.636-43.292, P=0.011) in hormone receptor-positive patients who were on adjuvant endocrine therapy. For breast cancer patients who did not receive adjuvant chemotherapy, those without infiltration of CD8-positive cells into tumor nests had a shorter overall survival (OS) than their counterparts with CD8-positive cell infiltration into tumor nests (Log-Rank, P=0.003). But OS of patients without infiltration of CD8-positive cells into tumor nests was significantly prolonged by adjuvant chemotherapy (Log-Rank, P=0.013) and paralleled that of patients with CD8-positive cell infiltration. Although OS was shorter in the tumor cell TGF-ß1 precursor (t-TGF-ß1-pre)-positive patients than in the negative patients in patients without receiving chemotherapy (P=0.053), OS of t-TGF-ß1-pre-positive patients was significantly prolonged by adjuvant chemotherapy (P=0.035) and was longer than that of t-TGF-ß1-pre-negative patients. Analysis showed that t-TGF-ß1-pre was an independent positive prognostic factor for DDFS (HR=0.392 95% CI=0.157-0.978, P=0.045) in patients who received adjuvant chemotherapy. This study suggested that density of interstitial CD8-positive lymphocytes was of prognostic value in hormone receptor-positive patients who received adjuvant endocrine therapy. Our study verified that adverse immunologic signatures consisting of absence of CD8-positive cells in tumor nests or expression of TGF-ß1 precursor in tumor cells in breast cancer were associated with worse prognosis and significantly improved long-term survival with adjuvant chemotherapy, respectively.

[Prognostic value of T lymphocytes infiltration in breast cancer].

OBJECTIVE: To assess and compare the prognostic role of tumor-infiltrating T lymphocytes in stage 1-3 breast cancer. METHODS: Paraffin sections were retrospectively collected from 130 cases of stage 1-3 breast cancer patients who received surgery between January 2000 and December 2002 in General Hospital of the People's Liberation Army. Immunohistochemistry was used to assess the density of tumor-infiltrating lymphocytes(TILs) that were positive of CD4 and CD8. These variables were evaluated for their association with histopathologic features along with overall survival(OS) , distant disease-free survival(DDFS) and disease-free survival(DFS) . RESULTS: Intraepithelial CD4+lymphocytes infiltration was an independent prognostic factor for DFS(HR=0.248, 95%CI=0.113-0.543, P=0.000) , DDFS(HR=0.361, 95%CI=0.157-0.830, P=0.017) , and OS(HR=0.297, 95%CI=0.119-0.741, P=0.009) in multifactor COX regression model. In hormone receptor negative group, mesenchymal CD8+lymphocytes and intraepithelial CD8+lymphocytes were independent prognostic factors for OS(HR=0.286, 95%CI=0.101-0.807, P=0.018) and DDFS(HR=0.293, 95%CI=0.104-0.825, P=0.020) , respectively. In hormone receptor positive group, mesenchymal CD8+lymphocytes and intraepithelial CD8+lymphocytes were independent prognostic factors for OS(HR=4.854, 95%CI=1.435-16.415, P=0.011) and DDFS(HR=10.493, 95%CI=1.226-89.795, P=0.032) respectively. Further analysis found that OS of hormone receptor positive patients with lower mesenchymal CD8+TILs was significantly proved by adjuvant endocrine therapy. CONCLUSIONS: In the current investigation, intraepithelial CD4+TILs demonstrated independent prognostic significance for survival. CD8+TILs were associated with better survival in hormone receptor negative patients but associated with worse survival in hormone receptor positive patients. The long-term clinical effects of adjuvant endocrine therapy is related with density of mesenchymal CD8+TILs and in turn affected prognostic value of mesenchymal CD8+TILs.

[Single oblique lumbar interbody fusion with robot-assisted posterior internal fixation for lumbar degenerative diseases].

OBJECTIVE: To investigate the efficacy of single oblique lumbar interbody fusion(OLIF) with robot-assisted posterior internal fixation for the treatment of lumbar degenerative diseases. METHODS: The clinical data of 67 patients with lumbar degenerative diseases treated from September 2019 to December 2020 was retrospectively analyzed. According to different surgical methods, the patients were divided into traditional group and robot group. The traditional group received traditional OLIF with posterior fluoroscopy percutaneous nail fixation, and the robot group received OLIF with robot-assisted posterior internal fixation. There were 33 patients in traditional group, including 13 males and 20 females, aged from 44 to 82 years old with an average of (59.7±9.1) years; and 34 cases in robot group, including 7 males and 27 females, aged from 45 to 81 years old with an average of(61.6±8.8) years. The operation time, fluoroscopy time, intraoperative blood loss, postoperative out of bed time and hospital stay were recorded. The visual analogue scale (VAS) of low back pain and Oswestry Disability Index(ODI) were compared before operation and 3 days, 3 months after operation between two groups. The accuracy of nail placement was evaluated by postoperative CT scan. RESULTS: Both groups of patients successfully completed the operation and were followed up for more than 3 months. The operation time, fluoroscopy time, intraoperative blood loss, postoperative out of bed time and hospital stay in traditional group were(299.85±15.79) min, (62.58±10.83) min, (118.33±10.80) ml, (2.5±0.7) d, (9.67±2.13) d;and robot group was(248.53±14.22) min, (19.47±3.51) min, (115.74±9.86) ml, (2.3±0.6) d, (9.44±1.93) d, respectively. The symptoms of postoperative low back pain, lower limb pain and numbness were significantly improved in all patients. The operation time and fluoroscopy time in robot group were significantly less than those of traditional group. There was no significant difference in intraoperative blood loss, postoperative out of bed time, hospital stay, VAS and ODI before and after operation (P>0.05). The accuracy of nail placement in robot group was 98.8% (2/160), which was higher than 89.9% (16/158) in traditional group. CONCLUSION: Treatment of lumbar degenerative diseases with single body position OLIF with robot-assisted posterior minimally invasive internal fixation has less operation time and fluoroscopy time, high nail placement accuracy and accurate surgical effect, which is worthy to be popularized in clinic.

Neurologic Injury in Complex Adult Spinal Deformity Surgery: Staged Multilevel Oblique Lumbar Interbody Fusion (MOLIF) Using Hyperlordotic Tantalum Cages and Posterior Fusion Versus Pedicle Subtraction Osteotomy (PSO).

STUDY DESIGN: A retrospective review of prospectively collected data. OBJECTIVE: The aim of this study was to determine the safety of MOLIF versus PSO. SUMMARY OF BACKGROUND DATA: Complex adult spinal deformity (CASD) represents a challenging cohort of patients. The Scoli-RISK-1 study has shown a 22.18% perioperative risk of neurological injury. Restoration of sagittal parameters is associated with good outcome in ASD. Pedicle subtraction osteotomies (PSO) is an important technique for sagittal balance in ASD but is associated with significant morbidity. The multilevel oblique lumbar interbody fusion (MOLIF) is an extensile approach from L1 to S1. METHODS: Single surgeon series from 2007 to 2015. Prospectively collected data. Scoli-RISK-1 criteria were refined to only include stiff or fused spines otherwise requiring a PSO. Roentograms were examined preoperatively and 2 year postoperatively. Primary outcome measure was the motor decline in American Spinal Injury Association (ASIA) at hospital discharge, 6 weeks, 6 months, and 2 years. Demographics, blood loss, operative time, spinopelvic parameters, and spinal cord monitoring (SCM) events. RESULTS: Sixty-eight consecutive patients were included in this study, with 34 patients in each Group. Group 1 (MOLIF) had a mean age 62.9 (45-81) and Group 2 (PSO) had a mean age of 66.76 years (47-79); 64.7% female versus PSO 76.5%; Body Mass Index (BMI) Group 1 (MOLIF) 28.05 and Group 2 (PSO) 27.17. Group 1 (MOLIF) perioperative neurological injury was 2.94% at discharge but resolved by 6 weeks. Group 2 (PSO) had five neurological deficits (14.7%) with no recovery by 2 years. There were four SCM events (SCM). In Group 1 (MOLIF), there was one event (2.94%) versus three events (8.88%) in Group 2 (PSO). CONCLUSION: Staged MOLIF avoids passing neurological structures or retraction of psoas and lumbar plexus. It is safer than PSO in CASD with stiff or fused spines with a lower perioperative neurological injury profile. MOLIF have less SCM events, blood loss, and number of levels fused. LEVEL OF EVIDENCE: 3.

Anti-Osteoporosis Medications Associated with Decreased Mortality after Hip Fracture.

OBJECTIVE: To study the effect of anti-osteoporosis therapies on mortality after hip fracture. METHODS: This retrospective study was carried out in the Second Affiliated Hospital of Fujian Medical University and enrolled 690 patients 50 years of age and older who were admitted with hip fractures between 2010 and 2015. The patients were followed in 2017: 690 patients aged was from 50 to 103 years. There were 456 women and 234 men. There were 335 patients with fractures of the femoral neck and 355 patients with intertrochanteric fractures of the femur. There were 444 (64.35%) patients who also had internal diseases. The Charlson comorbidity index was 0-6. The anti-osteoporosis medications were classified into no anti-osteoporosis medication, calcium + vitamin D supplementations, non-bisphosphonate medication, and bisphosphonate medication. The physicians followed the patients or family members by personal visit and telephone. Multivariable Cox regression analyses were done with known risk factors for mortality of hip fracture, such as gender, age, number of combined internal diseases, fracture type, place of residence, and Charlson comorbidity index, to show which anti-osteoporosis medications had significant effects on mortality after adjustment for these variables. RESULTS: Out of 690 patients with hip fractures, 149 patients received no anti-osteoporosis medication, 63 patients received calcium +vitamin D supplementations, 398 patients received non-bisphosphonate medication, and 80 patients received bisphosphonate medication. The patients were followed between 7 months and 52 months, with the average of 28.53 ± 9.75 months. A total of 166 patients died during the follow-up period. Of 166 deaths, 43 occurred within 3 months, 65 within 6 months, and 99 within 1 year after the hip fracture. In this study, fracture type, place of residence, and Charlson comorbidity index were not associated with the mortality, and the male gender, age > 75 years, and ≥ 2 combined internal diseases were the independent factors for deaths post-hip fracture. The cumulative mortality was 36.24% in the patients receiving no anti-osteoporosis medication. The hazard ratio for mortality after hip fracture with bisphosphonate medication, non-bisphosphonate medication, and calcium/vitamin D supplementation was 0.355 (95% CI, 0.194-0.648), 0.492 (95% CI, 0.347-0.699) and 0.616 (95% CI, 0.341-1.114), respectively, as compared with no anti-osteoporosis group. Bisphosphonate and non-bisphosphonate medications for osteoporosis were significantly associated with the reduction of cumulative mortality post-hip fracture (P < 0.01). CONCLUSIONS: Bisphosphonate and non-bisphosphonate medications for osteoporosis were significantly associated with decreased mortality after fragility hip fracture.

Effect of osteoprotegerin gene polymorphisms on the risk of cervical spondylotic myelopathy in a Chinese population.

OBJECTIVE: Cervical spondylotic myelopathy (CSM) is the most common cause of spinal cord dysfunction. Our study aims to explore the correlation of osteoprotegerin (OPG) gene polymorphisms and the risk factors and severity of CSM. PATIENTS AND METHODS: The peripheral blood samples from 494 CSM patients and 515 healthy individuals were collected for detecting the 950T/C, 1181G/C and 163A/G genotypes and genetic equilibrium of OPG in the CSM and control groups and analyzing the genotype distribution and allele frequency. The severity of CSM and the impaired segments were evaluated by the Japanese Orthopedic Association (JOA) scoring combined with cervical magnetic resonance imaging (MRI), in order to investigate the relations between the three genotypes of OPG promoter gene loci (950T/C, 163A/G and 1181G/C) and occurrence as well as severity of CSM. RESULTS: The risk rate of TC genotype carrier suffered from CSM was 0.46, of TT genotype carrier was 0.27. The risk rate of T allele carrier suffered from CSM was 0.37. In 950T/C single nucleotide polymorphism (SNP), patients with TC, TT and T genotypes had lower risk to suffer from CSM. CONCLUSION: Taken together, OPG 950T/C SNP protects against CSM, and it is correlated with the severity of CSM, providing a new idea for the prevention and treatment of CSM.

Low Grip Strength is a Strong Risk Factor of Osteoporosis in Postmenopausal Women.

OBJECTIVE: To investigate the effect of grip strength on bone mineral density (BMD) in postmenopausal women. Low BMD is related to risk of fracture and falling is the strongest factor for fragility fractures. Handgrip strength is a reliable indicator of muscle strength and muscle strength is associated with falling. METHODS: For the present study 120 women were divided into two groups: those ≤65 years and those >65 years. Serum 25 hydroxyvitamin D (25OHD), BMD, and handgrip strength were measured to observe the effect of age on 25OHD, grip strength, and BMD, as well as the effect of 25OHD on grip strength and BMD. The correlation between grip strength and BMD was investigated. RESULTS: In the 120 patients, 25OHD was 24.31 ± 8.29 ng/mL. There were 37 cases with 25OHD <20 ng/mL and 83 cases with 25 OHD ≥20 ng/mL. The patients with 25OHD <20 ng/mL had significantly lower femoral neck BMD, most of them with a T score ≤-2.5 (P < 0.05). BMD measurement showed 66 patients with femoral neck T ≤-2.5, 30 cases with total hip T ≤-2.5 and 90 cases with lumbar BMD T ≤-2.5. The maximum grip strength in the group is 22.28 ± 6.17 kg. There were 38 cases with the maximum grip strength <20 kg and 82 cases with the maximum grip strength ≥20 kg. Patients >65 years had lower 25OHD, lower maximum grip strength, and lower BMD. The osteoporosis risk in postmenopausal women with a maximum grip strength <20 kg and who were >65 years was significantly elevated. CONCLUSION: Handgrip strength and 25OHD decrease with aging in postmenopausal women. The patients with lower 25OHD level had significantly lower BMD of femoral neck. The patients with lower handgrip strength had significantly lower BMD of lumbar spine, femoral neck, and total hip. Grip strength measurement is the simplest muscle strength measurement method. Our study confirmed that low grip strength was correlated with low BMD and was a strong risk factor for osteoporosis in postmenopausal women.

[Surgical options for delayed osteoporotic vertebral collapse].

OBJECTIVE: To investigate the surgical options and clinical effects of delayed osteoporotic vertebral collapse. METHODS: From May 2010 to October 2014, 19 patients (20 vertebrae) with delayed osteoporotic vertebral collapse(Kümmell's disease) were enrolled in this study. There were 7 males and 12 females, aged from 65 to 87 years old with a mean of (73.5±5.62) years. According to Li staging system of Kümmell's disease, 3 cases were stage II, 13 cases (14 vertebrae) were stage III without spinal cord injury, 3 cases were stage III with spinal cord injury. Patients were respectively treated with percutaneous vertebroplasty(PVP) or percutaneous kyphoplasty(PKP) on the basis of the degree of postural reduction during operation. Injected cement volume, cement leakage, vertebral height restoration and local kyphotic reduction were observed. Visual analogue scale (VAS) and Oswestry Disability Index(ODI) were respectively used to assess the pain and function before and after operation. Frankel grade were used to evaluate neurological status. RESULTS: Seven vertebrae with satisfactory postural reduction were treated with PVP, 13 vertebrae with unsatisfactory postural reduction were treated with PKP, 3 patients with spinal cord injury were treated with decompression and posterior short segment fixation at the same time. All patients were followed up from 10 to 48 months with an average of 21.2 months. Cement leakage occurred in 4 cases with no symptom, 1 cases in PVP group and 3 cases in PKP group, there was no significant difference between two groups(P=0.561). The priming volume of cement was (6.40±0.94) ml in PVP group and (5.46±1.09) ml in PKP group (P>0.05). Three days after operation vs preoperation, the vertebral height restoration and kyphotic improvement was(31.71±11.35)%, (9.79±4.64)° in PVP group and (24.77±8.51)%, (8.15±2.97)° in PKP. There was no significant difference between two groups(P>0.05). Three days after operation, VAS of low back pain and ODI in all patients were improved than preoperative data(P<0.05), but there was no significant difference between two groups or between postoperative at 3 d and final follow up(P>0.05). Nerve function of 3 patients underwent decompression and fixation from Frankel D to E. CONCLUSIONS: According to Li staging system and the degree of introperative postural reduction, individualized surgical treatment for Kümmell's disease can obtain good clinical results. Bad postural reduction during operation maybe a risk factor of cement leakage.

Anterior and posterior fixation for delayed treatment of posterior atlantoaxial dislocation without fracture.

Posterior atlantoaxial dislocation (PAAD) without fracture of the odontoid process is a rare injury. Authors have variously reported closed or open reduction, followed by either anterior or posterior fixation, but there is no consensus on best treatment. We present a particularly unstable case of PAAD. Open reduction through a retropharyngeal approach with odontoidectomy was required for reduction. Anterior fixation with transarticular lag screws was required prior to posterior fixation with pedicle screws. Despite non-compliance with postoperative immobilisation, imaging at 20-month follow-up confirmed solid fusion. The patient is pain-free with a good range of movement of the neck and has returned to a manual job. Our case had a greater degree of instability than was previously reported, which necessitated 360° fixation. This is the first reported case of this treatment strategy, which provided a very stable fixation allowing fusion despite early movement and without causing undue stiffness.

[Expression and clinical significance of CD3, CD4 and COX-2 in non-small cell lung cancer].

AIM: To study expression and clinical significance of CD3, CD4 and COX-2 in non-small cell lung cancer (NSCLC). METHODS: Expression of COX-2, CD3 and CD4 was detected by immunohistochemical staining in 37 cases of NSCLC. The correlation of CD3, CD4, COX-2 expressions and overall survival(OS) was evaluated with spearman rank correlation analysis. RESULTS: The average surface density of CD3 and the average optical density of CD4 were significantly associated with OS of NSCLC patients after surgery (P<0.05). However, both two densities of COX-2 were correlated with survival time of patients (P>0.05). CONCLUSION: The expressions of CD3, CD4 were significantly associated with OS of NSCLC patients.

[Preparation of polyclonal and monoclonal antibodies against CRF and change of CRF in the sleep-deprived rat brain].

AIM: To produce polyclonal and monoclonal antibodies against corticotropin-releasing factor (CRF) and to study the changes of CRF in sleep-deprived rat brain with the antibodies acquired. METHODS: Commercial CRF was linked to bovine throglobulin (BTG) and bovine serum albumin (BSA) respectively to produce immunogen and embedding antigen. New Zealand rabbits and BALB/c mice were immunized with the BTG-CRF immunogen to produce polyclonal and monoclonal antibodies, respectively. The acquired antibodies were appraised with ELISA and immnohistochemical staining. The characterized antibodies were used to observe the changes of CRF in the rat brain 48 h after sleep deprivation. RESULTS: CRF polyclonal antibody and 9 clones of monoclonal antibodies were obtained with high titer, affinity and specificity. Among them, the polyclonal antibody and 2 monoclonal antibodies (1D10, 2F4) were excellent in immunocytochemical staining. The CRF-like immunoreactive substances were found more strongly expressed in the neurons of paraverticular hypothalamic nucleus (PVN), central subnucleus of amygdala (CeA), oval subnucleus of bed nucleus of stria terminalis (BNSTov), and nucleus of solitary tract (NTS) in sleep-deprived rat brain. While they were much weaker and even absent in the control. CONCLUSION: The polyclonal and monoclonal antibodies against CRF were successfully produced for immunocytochemical studies. The results indicate that CRF may play an important role in stress-responsive modulation during sleep deprivation. PVN, CeA and BNSTov are integral part of brain circuit related to the stress modulation.