Nonlinear Regression Modelling: A Primer with Applications and Caveats.

Use of nonlinear statistical methods and models are ubiquitous in scientific research. However, these methods may not be fully understood, and as demonstrated here, commonly-reported parameter p-values and confidence intervals may be inaccurate. The gentle introduction to nonlinear regression modelling and comprehensive illustrations given here provides applied researchers with the needed overview and tools to appreciate the nuances and breadth of these important methods. Since these methods build upon topics covered in first and second courses in applied statistics and predictive modelling, the target audience includes practitioners and students alike. To guide practitioners, we summarize, illustrate, develop, and extend nonlinear modelling methods, and underscore caveats of Wald statistics using basic illustrations and give key reasons for preferring likelihood methods. Parameter profiling in multiparameter models and exact or near-exact versus approximate likelihood methods are discussed and curvature measures are connected with the failure of the Wald approximations regularly used in statistical software. The discussion in the main paper has been kept at an introductory level and it can be covered on a first reading; additional details given in the Appendices can be worked through upon further study. The associated online Supplementary Information also provides the data and R computer code which can be easily adapted to aid researchers to fit nonlinear models to their data.

Intergroup Randomized Phase III Study of Postoperative Oxaliplatin, 5-Fluorouracil, and Leucovorin Versus Oxaliplatin, 5-Fluorouracil, Leucovorin, and Bevacizumab for Patients with Stage II or III Rectal Cancer Receiving Preoperative Chemoradiation: A Trial of the ECOG-ACRIN Research Group (E5204).

BACKGROUND: The addition of bevacizumab to chemotherapy improved outcomes for patients with metastatic colon cancer. E5204 was designed to test whether the addition of bevacizumab to mFOLFOX6, following neoadjuvant chemoradiation and definitive surgery, could improve overall survival (OS) in patients with stage II/III adenocarcinoma of the rectum. SUBJECTS, MATERIALS, AND METHODS: Patients with stage II/III rectal cancer who had completed neoadjuvant 5-fluorouracil-based chemoradiation and had undergone complete resection were enrolled. Patients were randomized to mFOLFOX6 (Arm A) or mFOLFOX6 with bevacizumab (Arm B) administered every 2 weeks for 12 cycles. RESULTS: E5204 registered only 355 patients (17% of planned accrual goal) as it was terminated prematurely owing to poor accrual. At a median follow-up of 72 months, there was no difference in 5-year overall survival (88.3% vs. 83.7%) or 5-year disease-free survival (71.2% vs. 76.5%) between the two arms. The rate of treatment-related grade ≥ 3 adverse events (AEs) was 68.8% on Arm A and 70.7% on Arm B. Arm B had a higher proportion of patients who discontinued therapy early as a result of AEs and patient withdrawal than did Arm A (32.4% vs. 21.5%, p = .029).The most common grade 3-4 treatment-related AEs were neutropenia, leukopenia, neuropathy, diarrhea (without prior colostomy), and fatigue. CONCLUSION: At 17% of its planned accrual, E5204 did not meet its primary endpoint. The addition of bevacizumab to FOLFOX6 in the adjuvant setting did not significantly improve OS in patients with stage II/III rectal cancer. IMPLICATIONS FOR PRACTICE: At 17% of its planned accrual, E5204 was terminated early owing to poor accrual. At a median follow-up of 72 months, there was no significant difference in 5-year overall survival (88.3% vs. 83.7%) or in 5-year disease-free survival (71.2% vs. 76.5%) between the two arms. Despite significant advances in the treatment of rectal cancer, especially in improving local control rates, the risk of distant metastases and the need to further improve quality of life remain a challenge. Strategies combining novel agents with chemoradiation to improve both distant and local control are needed.

Analysis of Coronavirus Temperature-Sensitive Mutants Reveals an Interplay between the Macrodomain and Papain-Like Protease Impacting Replication and Pathogenesis.

Analysis of temperature-sensitive (ts) mutant viruses is a classic method allowing researchers to identify genetic loci involved in viral replication and pathogenesis. Here, we report genetic analysis of a ts strain of mouse hepatitis virus (MHV), tsNC11, focusing on the role of mutations in the macrodomain (MAC) and the papain-like protease 2 (PLP2) domain of nonstructural protein 3 (nsp3), a component of the viral replication complex. Using MHV reverse genetics, we generated a series of mutant viruses to define the contributions of macrodomain- and PLP2-specific mutations to the ts phenotype. Viral replication kinetics and efficiency-of-plating analysis performed at permissive and nonpermissive temperatures revealed that changes in the macrodomain alone were both necessary and sufficient for the ts phenotype. Interestingly, mutations in the PLP2 domain were not responsible for the temperature sensitivity but did reduce the frequency of reversion of macrodomain mutants. Coimmunoprecipitation studies are consistent with an interaction between the macrodomain and PLP2. Expression studies of the macrodomain-PLP2 portion of nsp3 indicate that the ts mutations enhance proteasome-mediated degradation of the protein. Furthermore, we found that during virus infection, the replicase proteins containing the MAC and PLP2 mutations were more rapidly degraded at the nonpermissive temperature than were the wild-type proteins. Importantly, we show that the macrodomain and PLP2 mutant viruses trigger production of type I interferon in vitro and are attenuated in mice, further highlighting the importance of the macrodomain-PLP2 interplay in viral pathogenesis.IMPORTANCE Coronaviruses (CoVs) are emerging human and veterinary pathogens with pandemic potential. Despite the established and predicted threat these viruses pose to human health, there are currently no approved countermeasures to control infections with these viruses in humans. Viral macrodomains, enzymes that remove posttranslational ADP-ribosylation of proteins, and viral multifunctional papain-like proteases, enzymes that cleave polyproteins and remove polyubiquitin chains via deubiquitinating activity, are two important virulence factors. Here, we reveal an unanticipated interplay between the macrodomain and the PLP2 domain that is important for replication and antagonizing the host innate immune response. Targeting the interaction of these enzymes may provide new therapeutic opportunities to treat CoV disease.

Subventricular zone neural precursor cell responses after traumatic brain injury and binge alcohol in male rats.

Traumatic brain injury (TBI) is a major cause of disability worldwide. Additionally, many TBI patients are intoxicated with alcohol at the time of injury, but the impact of acute intoxication on recovery from brain injury is not well understood. We have previously found that binge alcohol prior to TBI impairs spontaneous functional sensorimotor recovery. However, whether alcohol administration in this setting affects reactive neurogenesis after TBI is not known. This study, therefore, sought to determine the short- and long-term effects of pre-TBI binge alcohol on neural precursor cell responses in the subventricular zone (SVZ) following brain injury in male rats. We found that TBI alone significantly increased proliferation in the SVZ as early as 24 hr after injury. Surprisingly, binge alcohol alone also significantly increased proliferation in the SVZ after 24 hr. However, a combined binge alcohol and TBI regimen resulted in decreased TBI-induced proliferation in the SVZ at 24 hr and 1 week post-TBI. Furthermore, at 6 weeks after TBI, binge alcohol administered at the time of TBI significantly decreased the TBI-induced neuroblast response in the SVZ and the rostral migratory stream (RMS). The results from this study suggest that pre-TBI binge alcohol negatively impacts reparative processes in the brain by decreasing short-term neural precursor cell proliferative responses as well as long-term neuroblasts in the SVZ and RMS.

Influence of Rehabilitation Medicine Residency Training in Performing Chemodenervation in Children with Cerebral Palsy in Thailand.

Background: Chemodenervation (CD) involves injecting drugs such as phenol, botulinum toxin, or alcohol to reduce muscle spasticity. However, they interfere with daily activities of children with cerebral palsy (CP). Rehabilitation residency training in Thailand currently requires performing a minimum of five CD procedures. However, the effect of this policy on post-training practice is unknown. Objective: To explore the influence of CD training during residency on post-training clinical practice and their current use of it in treating CP patients. Material and Method: The questionnaires were sent to 431 Thai physiatrists nationwide by both electronic and postal mails. The responses were collected within a three-month period. Results: Of 116 (27%) respondents with usable questionnaires, 85 (73%) were trained during their residency to perform CD and 46 (40%) performed it in their practice. Those trained to perform CD were more likely in their subsequent practice to do so (p = 0.0140), and younger age was associated with performing it (p = 0.0055). The number of CD procedures performed during residency correlated directly with reported confidence in performing the procedure in later practice (p<0.0001). The most common reasons for not performing CD were few CP cases in their care, and unavailable equipment or injection agent. Conclusion: Although only a cross-sectional study, the findings suggest that increasing the number of CD procedures required in rehabilitation residency may increase the use of CD to benefit CP patients in future clinical practice.

Human Leukocyte Antigen Markers for Distinguishing Pustular Psoriasis and Adult-Onset Immunodeficiency with Pustular Reaction.

Pustular skin diseases, with pustular psoriasis (PP) being the prototype, are immune-mediated diseases characterized by the presence of multiple pustules, resulting from neutrophil accumulation in the layer of epidermis. Sterile skin pustular eruption, like PP, is also observed in 20-30% of patients with adult-onset immunodeficiency syndrome (AOID) and anti-interferon γ autoantibodies (IFN-γ), leading to challenges in classification and diagnosis. While the mechanism underlying this similar phenotype remains unknown, genetic factors in relation to the immune system are suspected of playing an important role. Here, the association between human leukocyte antigen (HLA) genes, which play essential roles in antigen presentation, contributing to immune response, and the presence of skin pustules in AOID and PP was revealed. HLA genotyping of 41 patients from multiple centers in Thailand who presented with multiple sterile skin pustules (17 AOID patients and 24 PP patients) was conducted using a next-generation-sequencing-based approach. In comparison to healthy controls, HLA-B*13:01 (OR = 3.825, 95%CI: 2.08-7.035), C*03:04 (OR = 3.665, 95%CI: 2.102-6.39), and DQB1*05:02 (OR = 2.134, 95%CI: 1.326-3.434) were significantly associated with the group of aforementioned conditions having sterile cutaneous pustules, suggesting a common genetic-related mechanism. We found that DPB1*05:01 (OR = 3.851, p = 0.008) and DRB1*15:02 (OR = 3.195, p = 0.033) have a significant association with pustular reaction in AOID patients, with PP patients used as a control. A variant in the DRB1 gene, rs17885482 (OR = 9.073, p = 0.005), was observed to be a risk factor for PP when using AOID patients who had pustular reactions as a control group. DPB1*05:01 and DRB1*15:02 alleles, as well as the rs17885482 variant in the DRB1 gene, were proposed as novel biomarkers to differentiate PP and AOID patients who first present with multiple sterile skin pustules without known documented underlying conditions.

Postmortem Alteration of Purine Metabolism in Coronary Artery Disease.

A new approach for assisting in the diagnosis of coronary artery disease (CAD) as a cause of death is essential in cases where complete autopsy examinations are not feasible. The purine pathway has been associated with CAD patients, but the understanding of this pathway in postmortem changes needs to be explored. This study investigated the levels of blood purine metabolites in CAD after death. Heart blood samples (n = 60) were collected and divided into CAD (n = 23) and control groups (n = 37). Purine metabolites were measured via proton nuclear magnetic resonance. Guanosine triphosphate (GTP), nicotinamide adenine dinucleotide (NAD), and xanthine levels significantly decreased (p < 0.05); conversely, adenine and deoxyribose 5-phosphate levels significantly increased (p < 0.05) in the CAD group compared to the control group. Decreasing xanthine levels may serve as a marker for predicting the cause of death in CAD (AUC = 0.7). Our findings suggest that the purine pathway was interrupted by physiological processes after death, causing the metabolism of the deceased to differ from that of the living. Additionally, xanthine levels should be studied further to better understand their relationship with CAD and used as a biomarker for CAD diagnosis under decomposition and skeletonization settings.

Categorization of Cytochrome P4502D6 Activity Score by Urinary Amphetamine/Methamphetamine Ratios.

Methamphetamine (MA) level in urine has been used for judgment in MA consumption. Metabolism and intoxication of MA are correlated with the activity of cytochrome P450 2D6 (CYP2D6). The activity score (AS) is a potential tool for predicting exposure and personalized dose of drugs metabolized by CYP2D6. Prediction of the CYP2D6 activity score might be described as MA intoxication. The objective of this study was to categorize the CYP2D6 activity score using the urinary amphetamine (AM)/MA ratio. Urine samples (n = 23,258) were collected. The levels of MA and AM were determined by a gas chromatography-nitrogen-phosphorus detector. The log AS was calculated by an AM/MA ratio and classified into four groups following the percentile position: lower than the 2.5th, the 2.5th-the 50th, the 50th-97.5th, and greater than the 97.5th percentile, respectively. The AS value for males presented was less than 0.024, 0.024-0.141, 0.141-0.836, and greater than 0.836. Meanwhile, the AS values were revealed to be lower than 0.023, 0.023-0.148, 0.148-0.850, and higher than 0.850 for females. The AS value of CYP2D6 can be applied to describe the toxicity of MA in forensic crime scenes and relapse behavior.

Efficient experimental design for dose response modelling.

The logit binomial logistic dose response model is commonly used in applied research to model binary outcomes as a function of the dose or concentration of a substance. This model is easily tailored to assess the relative potency of two substances. Consequently, in instances where two such dose response curves are parallel so one substance can be viewed as a dilution of the other, the degree of that dilution is captured in the relative potency model parameter. It is incumbent that experimental researchers working in fields including biomedicine, environmental science, toxicology and applied sciences choose efficient experimental designs to run their studies to both fit their dose response curves and to garner important information regarding drug or substance potency. This article provides far-reaching practical design strategies for dose response model fitting and estimation of relative potency using key illustrations. These results are subsequently extended here to handle situations where the assessment of parallelism and the proper dose-scale are also of interest. Conclusions and recommended strategies are supported by both theoretical and simulation results.

Detecting SARS-CoV-2 3CLpro expression and activity using a polyclonal antiserum and a luciferase-based biosensor.

The need to stem the current outbreak of SARS-CoV-2 responsible for COVID-19 is driving the search for inhibitors that will block coronavirus replication and pathogenesis. The coronavirus 3C-like protease (3CLpro) encoded in the replicase polyprotein is an attractive target for antiviral drug development because protease activity is required for generating a functional replication complex. Reagents that can be used to screen for protease inhibitors and for identifying the replicase products of SARS-CoV-2 are urgently needed. Here we describe a luminescence-based biosensor assay for evaluating small molecule inhibitors of SARS-CoV-2 3CLpro/main protease. We also document that a polyclonal rabbit antiserum developed against SARS-CoV 3CLpro cross reacts with the highly conserved 3CLpro of SARS-CoV-2. These reagents will facilitate the pre-clinical evaluation of SARS-CoV-2 protease inhibitors.

Improved Functional Outcome After Peripheral Nerve Stimulation of the Impaired Forelimb Post-stroke.

Lack of blood flow to the brain, i.e., ischemic stroke, results in loss of nerve cells and therefore loss of function in the effected brain regions. There is no effective treatment to improve lost function except restoring blood flow within the first several hours. Rehabilitation strategies are widely used with limited success. The purpose of this study was to examine the effect of electrical stimulation on the impaired upper extremity to improve functional recovery after stroke. We developed a rodent model using an electrode cuff implant onto a single peripheral nerve (median nerve) of the paretic forelimb and applied daily electrical stimulation. The skilled forelimb reaching test was used to evaluate functional outcome after stroke and electrical stimulation. Anterograde axonal tracing from layer V pyramidal neurons with biotinylated dextran amine was done to evaluate the formation of new neuronal connections from the contralesional cortex to the deafferented spinal cord. Rats receiving electrical stimulation on the median nerve showed significant improvement in the skilled forelimb reaching test in comparison with stroke only and stroke with sham stimulation. Rats that received electrical stimulation also exhibited significant improvement in the latency to initiate adhesive removal from the impaired forelimb, indicating better sensory recovery. Furthermore, axonal tracing analysis showed a significant higher midline fiber crossing index in the cervical spinal cord of rats receiving electrical stimulation. Our results indicate that direct peripheral nerve stimulation leads to improved sensorimotor recovery in the stroke-impaired forelimb, and may be a useful approach to improve post-stroke deficits in human patients.

Dentate Gyrus Proliferative Responses After Traumatic Brain Injury and Binge Alcohol in Adult Rats.

BACKGROUND: Traumatic brain injury is a significant public health issue that results in serious disability in survivors. Traumatic brain injury patients are often intoxicated with alcohol when admitted to the hospital; however, it is not clear how acute intoxication affects recovery from a traumatic brain injury. Our group has previously shown that binge alcohol prior to traumatic brain injury resulted in long-term impairment in a fine sensorimotor task that was correlated with a decreased proliferative and neuroblast response from the subventricular zone. However, whether binge alcohol prior to traumatic brain injury affects the proliferative response in the hippocampal dentate gyrus is not yet known. METHODS: Male rats underwent binge alcohol (3 g/kg/day) by gastric gavage for 3 days prior to traumatic brain injury. Cell proliferation was labeled by BrdU injections following traumatic brain injury. Stereological quantification and immunofluorescence confocal analysis of BrdU+ cells in the hippocampal dorsal dentate gyrus was performed at 24 hours, 1 week and 6 weeks post traumatic brain injury. RESULTS: We found that either traumatic brain injury alone or binge alcohol alone significantly increased dentate gyrus proliferation at 24 hours and 1 week. However, a combined binge alcohol and traumatic brain injury regimen resulted in decreased dentate gyrus proliferation at 24 hours post-traumatic brain injury. At the 6 week time point, binge alcohol overall reduced the number of BrdU+ cells. Furthermore, more BrdU+ cells were found in the dentate hilar region of alcohol traumatic brain injury compared to vehicle traumatic brain injury groups. The location and double-labeling of these mismigrated BrdU+ cells was consistent with hilar ectopic granule cells. CONCLUSION: The results from this study showed that pre-traumatic brain injury binge alcohol impacts the injury-induced proliferative response in the dentate gyrus in the short-term and may affect the distribution of newly generated cells in the dentate gyrus in the long-term.

A Three-Arm Randomized Phase II Study of Bendamustine/Rituximab with Bortezomib Induction or Lenalidomide Continuation in Untreated Follicular Lymphoma: ECOG-ACRIN E2408.

PURPOSE: We sought to improve upon frontline bendamustine/rituximab (BR) induction therapy followed by rituximab maintenance in untreated high-risk follicular lymphoma (FL). PATIENTS AND METHODS: Patients were randomized to BR induction followed by 2-year rituximab maintenance (BR-R), BR with bortezomib and rituximab maintenance (BVR-R), or BR followed by lenalidomide (1 year) with rituximab maintenance (BR-LR). Dual primary objectives were complete remission (CR) rate and 1-year disease-free survival (DFS); 289 patients enrolled (NCT01216683). RESULTS: For induction, 92%, 87%, and 86% of patients randomized to BR-R, BVR-R, or BR-LR received six cycles, respectively. CR rate with BR versus BVR induction was 62% versus 75%, respectively (P = 0.04). One-year DFS rates with BR-R versus BR-LR were 85% versus 67%, respectively (P = 0.0009). This was due to an imbalance in CR rates post-BR induction and discontinuation due to adverse events (AEs). The most common grade 3-4 AEs for BVR versus BR were neutropenia and sensory neuropathy (12% vs <1%); 83% of the latter occurred with intravenous bortezomib. The most common grade 3-4 AEs related to LR versus rituximab maintenance were neutropenia 66% versus 21%, respectively (P < 0.0001), and febrile neutropenia 10% versus 2%, respectively (P = 0.05). The overall treatment-related mortality was 1.4%. With 5-year median follow-up, 3-year PFS rates for BR-R, BVR-R, and BR-LR were 77%, 82%, and 76%, respectively (P = 0.36) with OS rates of 87%, 90%, and 84%, respectively (P = 0.79). For prognostication, CR rate and POD-24 were associated with survival. CONCLUSIONS: Altogether, neither bortezomib added to BR induction nor lenalidomide added to rituximab maintenance immediately post-BR induction is recommended in untreated FL.

Improved functional outcome after chronic stroke with delayed anti-Nogo-A therapy: A clinically relevant intention-to-treat analysis.

Many preclinical treatment strategies for stroke have failed when tested in human trials. Although the reasons for these translation failures are multifactorial, one potential concern is the statistical analysis of the preclinical data. One way to rigorously evaluate new therapies is to use an intention-to-treat analysis in preclinical studies. Therefore, in this study, we set out to evaluate the treatment efficacy of a potential clinically relevant therapeutic agent for stroke, i.e., anti-Nogo-A immunotherapy, using an intention-to-treat analysis. Adult rats were trained on the skilled forelimb reaching task and subsequently underwent an ischemic stroke. Nine weeks later, the rats either received intracerebroventricular anti-Nogo-A antibody, control antibody, or no treatment. Skilled reaching performance was assessed by a non-linear model using both an intention-to-treat and per-protocol analysis. Following testing, dendritic complexity was evaluated in the contralesional and perilesional sensorimotor cortex. Both intention-to-treat and per-protocol analysis showed that anti-Nogo-A immunotherapy resulted in statistically significant improved recovery on the skilled forelimb reaching task, although treatment effect was less (though statistically significant) in the intention-to-treat group. Improved functional performance was not shown to be associated with dendritic changes. In conclusion, this study provides evidence for the importance of using intention-to-treat paradigms in testing preclinical therapeutic strategies.

Stratification of ovarian tumor pathology by expression of programmed cell death-1 (PD-1) and PD-ligand- 1 (PD-L1) in ovarian cancer.

BACKGROUND: Ovarian cancer is the major cause of death among gynecologic cancers with 75% of patients diagnosed with advanced disease, and only 20% of these patients having a survival duration of five years. Treatments blocking immune checkpoint molecules, programmed cell death (PD-1) or its ligand PD-ligand- I (PD-L1) have produced a beneficial and prolonged effect in a subgroup of these patients. However, there is debate in the literature concerning the prognostic value of the expression of these molecules in tumors, with immunotherapy responsiveness, and survival. We evaluated the immune landscape of the ovarian tumor microenvironment of patients, by measuring the impact of the expression of tumor PD-1, PD-L1 and infiltrating lymphocytes on stage and grade of tumors and survival, in a cohort of 55 patients with gynecologic malignancies. Most patients under study were diagnosed with advanced disease ovarian cancer. RESULTS: Our studies revealed that a low density of PD-1 and of PD-L1 expressing cells in tumor tissue were significantly associated with advanced disease (P = 0.028 and P = 0.033, respectively). Moreover, PD-L1 was expressed significantly more often in high grade tumors (41.5%) than in low grade tumors of patients (7.7%) (P = 0.040). The presence of CD3 or of FoxP3 infiltrating cells with PD-L1 in patient tumors did not impact the significance of the association of PD-L1 with high grade tumors (P = 0.040), and our analyses did not show an association between the presence of PD-1 or PD-L1 and survival. CONCLUSIONS: We conclude that a subgroup of advanced disease ovarian cancer patients with high grade tumors, expressing PD-L1, may be prime candidates for immunotherapy targeting PD-1 signaling.

Phase II trial of subcutaneous recombinant human interleukin 11 with subcutaneous recombinant human granulocyte-macrophage colony stimulating factor in patients with acute myeloid leukemia (AML) receiving high-dose cytarabine during induction: ECOG 3997.

Randomized trials of substituting high-dose cytarabine (HiDAC) for standard dose cytarabine (SDAC) during induction therapy for newly diagnosed AML have not demonstrated an improvement in the complete remission (CR) rate. Phase II trials of the scheduled administration of HiDAC after SDAC suggest an improved outcome. The hematological complications of intensification are considerable. GM-CSF after chemotherapy improved the survival of older patients in a randomized trial. Recombinant human interleukin 11, a thrombopoietic cytokine, reduced the incidence of chemotherapy-induced thrombocytopenia in patients with solid tumors. Therefore, 34 patients were treated, with newly diagnosed AML less than 56 years of age, with daunorubicin 45 mg/m2 on days 1-3, cytarabine 100mg/m2 days 1-7 and cytarabine 2g/m2 for 12 h on days 8-10 (7+3+3). rhIL-11 (50 microg/kg/day,) and GM-CSF (250 microg/kg/day) were administered subcutaneously from day 11 until recovery. The complete remission rate was 59% (90% C.I. 43-73%). The median time to recovery of neutrophils to >500 and platelets to > or =20,000 microl(-1) was 27 days (95% C.I. 27-30 days) and 25 days (95% C.I. 24-29 days), respectively. The trial does not confirm the high CR rate observed in phase II trials, despite optimal supportive care.

Axonal plasticity is associated with motor recovery following amphetamine treatment combined with rehabilitation after brain injury in the adult rat.

Clinical and laboratory studies have suggested that amphetamine treatment when paired with rehabilitation results in improved recovery of function after stroke or traumatic brain injury. In the present study, we investigated whether new anatomical pathways developed in association with improved motor function after brain damage and amphetamine treatment linked with rehabilitation. Following a unilateral sensorimotor cortex lesion in the adult rat, amphetamine (2 mg/kg) was administered in conjunction with physiotherapy sessions on postoperative days two and five. Physiotherapy was continued twice daily for the first 3 weeks after injury, and then once daily until week six. Performance on skilled forelimb reaching and ladder rung walking was used to assess motor improvement. Our results show that animals with sensorimotor cortical lesions receiving amphetamine treatment linked with rehabilitation had significant improvement in both tasks. Neuroanatomical tracing of efferent pathways from the opposite, non-damaged cortex resulted in the novel finding that amphetamine treatment linked with rehabilitation, significantly increased axonal growth in the deafferented basilar pontine nuclei. These results support the notion that pharmacological interventions paired with rehabilitation can enhance neuronal plasticity and thereby improve functional recovery after CNS injury.

Acute toxicity of binary-metal mixtures of copper, zinc, and nickel to Pimephales promelas: Evidence of more-than-additive effect.

Metal mixture toxicity has been studied for decades. However, the results are not consistent, and thus ecological risk assessment and regulation of mixtures has been difficult. The objective of the present study was to use a systematic experimental design to characterize the toxicity of binary-metal mixture of Cu, Zn, and Ni to Pimephales promelas, typically to determine whether the effect of these binary-metal mixtures on P. promelas is more-than-additive. Standard 96-h toxicity tests were conducted with larval P. promelas based on US Environmental and Protection Agency methods to determine metal mixture effects. All experiments were conducted in synthetic moderately hard water with no addition of dissolved organic matter. Three different effect analysis approaches, the MixTox model, the Finney model, and the toxic unit method, were used for comparison. The results indicate that the toxicity of Cu+Zn, Cu+Ni, and Zn+Ni mixtures to P. promelas was more-than-additive. Among the 3 mixtures, the effect of the Cu+Ni mixture was the most profound. The results of the present study are useful for applications to models such as the metal mixture biotic ligand model. More research should be conducted to determine the mechanisms of acute and chronic toxicity of metal mixtures.

Anti-Nogo-A Immunotherapy Does Not Alter Hippocampal Neurogenesis after Stroke in Adult Rats.

Ischemic stroke is a leading cause of adult disability, including cognitive impairment. Our laboratory has previously shown that treatment with function-blocking antibodies against the neurite growth inhibitory protein Nogo-A promotes functional recovery after stroke in adult and aged rats, including enhancing spatial memory performance, for which the hippocampus is critically important. Since spatial memory has been linked to hippocampal neurogenesis, we investigated whether anti-Nogo-A treatment increases hippocampal neurogenesis after stroke. Adult rats were subject to permanent middle cerebral artery occlusion followed 1 week later by 2 weeks of antibody treatment. Cellular proliferation in the dentate gyrus was quantified at the end of treatment, and the number of newborn neurons was determined at 8 weeks post-stroke. Treatment with both anti-Nogo-A and control antibodies stimulated the accumulation of new microglia/macrophages in the dentate granule cell layer, but neither treatment increased cellular proliferation or the number of newborn neurons above stroke-only levels. These results suggest that anti-Nogo-A immunotherapy does not increase post-stroke hippocampal neurogenesis.

Delayed treatment with monoclonal antibody IN-1 1 week after stroke results in recovery of function and corticorubral plasticity in adult rats.

Neuronal death due to ischemic stroke results in permanent deficits in sensory, language, and motor functions. The growth-restrictive environment of the adult central nervous system (CNS) is an obstacle to functional recovery after stroke and other CNS injuries. In this regard, Nogo-A is a potent neurite growth-inhibitory protein known to restrict neuronal plasticity in adults. Previously, we have found that treatment with monoclonal antibody (mAb) IN-1 to neutralize Nogo-A immediately after stroke enhanced motor cortico-efferent plasticity and recovery of skilled forelimb function in rats. However, immediate treatment for stroke is often not clinically feasible. Thus, the present study was undertaken to determine whether cortico-efferent plasticity and functional recovery would occur if treatment with mAb IN-1 was delayed 1 week after stroke. Adult rats were trained on a forelimb-reaching task, and the middle cerebral artery was occluded to induce focal cerebral ischemia to the forelimb sensorimotor cortex. After 1 week, animals received mAb IN-1 treatment, control antibody, or no treatment, and were tested for 9 more weeks. To assess cortico-efferent plasticity, the sensorimotor cortex opposite the stroke lesion was injected with an anterograde neuroanatomical tracer. Behavioral analysis demonstrated a recovery of skilled forelimb function, and anatomical studies revealed neuroplasticity at the level of the red nucleus in animals treated with mAb IN-1, thus demonstrating the efficacy of this treatment even if administered 1 week after stroke.