A practical guide to data management and sharing for biomedical laboratory researchers.

Effective data management and sharing have become increasingly crucial in biomedical research; however, many laboratory researchers lack the necessary tools and knowledge to address this challenge. This article provides an introductory guide into research data management (RDM), and the importance of FAIR (Findable, Accessible, Interoperable, and Reusable) data-sharing principles for laboratory researchers produced by practicing scientists. We explore the advantages of implementing organized data management strategies and introduce key concepts such as data standards, data documentation, and the distinction between machine and human-readable data formats. Furthermore, we offer practical guidance for creating a data management plan and establishing efficient data workflows within the laboratory setting, suitable for labs of all sizes. This includes an examination of requirements analysis, the development of a data dictionary for routine data elements, the implementation of unique subject identifiers, and the formulation of standard operating procedures (SOPs) for seamless data flow. To aid researchers in implementing these practices, we present a simple organizational system as an illustrative example, which can be tailored to suit individual needs and research requirements. By presenting a user-friendly approach, this guide serves as an introduction to the field of RDM and offers practical tips to help researchers effortlessly meet the common data management and sharing mandates rapidly becoming prevalent in biomedical research.

Training following unilateral cervical spinal cord injury in rats affects the contralesional forelimb.

Rehabilitative training is an essential component of current therapeutic strategies for spinal cord injured individuals. However, there are still various open questions that need to be answered in order to optimize training strategies. For example, why can animals trained in a single task perform worse compared to untrained animals when tested in untrained tasks. Such results suggest a potential competition among motor tasks over spared neuronal circuitry. Whether training induced competition for neuronal circuitry may also exist between injured and spared circuitries of the ipsi- and contralesional extremity is currently unknown. Here we investigated whether training restricted to the frontlimb ipsilateral to cervical spinal injury (IF) can impact motor performance of the contralesional frontlimb (CF) in a rat model of cervical SCI. We compared CF performance following general motor training of all limbs (horizontal ladder), following specific training of the IF (pellet reaching), as well as following a combination of both training paradigms. Our findings indicate that adding ipsilateral side-specific training to general training can negatively impact performance of the CF, without resulting in any improvement of performance of the IF. In conclusion, our results emphasize that important decisions have to be made when designing rehabilitative training strategies, ideally taking into account more than the primarily affected extremity.

Locomotion after spinal cord injury depends on constitutive activity in serotonin receptors.

Following spinal cord injury (SCI) neurons caudal to the injury are capable of rhythmic locomotor-related activity that can form the basis for substantial functional recovery of stepping despite the loss of crucial brain stem-derived neuromodulators like serotonin (5-HT). Here we investigated the contribution of constitutive 5-HT(2) receptor activity (activity in the absence of 5-HT) to locomotion after SCI. We used a staggered hemisection injury model in rats to study this because these rats showed a robust recovery of locomotor function and yet a loss of most descending axons. Immunolabeling for 5-HT showed little remaining 5-HT below the injury, and locomotor ability was not correlated with the amount of residual 5-HT. Furthermore, blocking 5-HT(2) receptors with an intrathecal (IT) application of the neutral antagonist SB242084 did not affect locomotion (locomotor score and kinematics were unaffected), further indicating that residual 5-HT below the injury did not contribute to generation of locomotion. As a positive control, we found that the same application of SB242084 completely antagonized the muscle activity induced by exogenous application of the 5-HT(2) receptor agonists alpha-methyl-5-HT (IT). In contrast, blocking constitutive 5-HT(2) receptor activity with the potent inverse agonist SB206553 (IT) severely impaired stepping as assessed with kinematic recordings, eliminating most hindlimb weight support and overall reducing the locomotor score in both hind legs. However, even in the most severely impaired animals, rhythmic sweeping movements of the hindlimb feet were still visible during forelimb locomotion, suggesting that SB206553 did not completely eliminate locomotor drive to the motoneurons or motoneuron excitability. The same application of SB206553 had no affect on stepping in normal rats. Thus while normal rats can compensate for loss of 5-HT(2) receptor activity, after severe spinal cord injury rats require constitutive activity in these 5-HT(2) receptors to produce locomotion.

Transplantation and repair: combined cell implantation and chondroitinase delivery prevents deterioration of bladder function in rats with complete spinal cord injury.

STUDY DESIGN: Additional examination. In this study, we report changes in bladder function after a combined treatment that was designed to study axonal regeneration after complete spinal cord injury (SCI) in rats. OBJECTIVES: To report effects on bladder function following the administration of a combined treatment for complete SCI. SETTING: University of Alberta, Faculty of Rehabilitation Medicine, Edmonton, Canada. METHODS: Eight rats received Schwann cells in Matrigel-filled guidance channels, olfactory ensheathing glia and chondroitinase ABC at the lesion site following complete thoracic SCI. Controls (n=7) received Matrigel only. Daily bladder examinations were performed. Analysis of bladder size, wall thickness, actin and collagen type III was performed after 14 weeks. RESULTS: Following SCI, both groups regained bladder voiding after 3 weeks. However, 2 weeks later, incontinence was observed in all untreated rats and two treated rats. Post-mortem examination of bladders revealed enlarged bladder sizes. Thicker bladder walls were found in untreated rats, which were composed of disorganized bundles of smooth muscle fibers surrounded by high amounts of collagen (type III). CONCLUSION: We show that the combined treatment prevents collagen deposition in bladder walls and maintains the rat's ability to void efficiently. Although the mechanism responsible for this improvement is unclear, our study shows that the present combinatory therapy can influence bladder function, thus expanding their utility as a broad reparative approach for SCI.

BDNF promotes connections of corticospinal neurons onto spared descending interneurons in spinal cord injured rats.

Although regeneration of injured axons is inhibited within the adult CNS, moderate recovery can be found in patients and animals with incomplete spinal cord injury (SCI). This can be partly attributed to sprouting of spared and injured axons, rostral and caudal to the lesion, respectively. Recently, it has been reported that following a thoracic SCI such sprouting can result in indirect reconnections of the lesioned axons to caudal targets via propriospinal interneurons (PrI). Here, we attempted to further promote this spontaneous repair mechanism by applying the neurotrophic factor BDNF (brain-derived neurotrophic factor), in the vicinity of the cell bodies of lesioned corticospinal neurons or NT-3, intrathecally to the cervical spinal cord. We performed a dorsal over-hemisection at the thoracic spinal cord sparing only the left ventrolateral quadrant. This type of lesion did not promote sprouting of injured corticospinal axons or re-routing via commissural PrI. Also, in rats that received NT-3 at the cervical enlargement, no increase in sprouting was found. However, animals receiving BDNF at the cell bodies of lesioned corticospinal neurons showed a significant increase in collateral sprouting and in the number of contacts with PrI. This was not observed when BDNF was administered to unlesioned animals. Although no statistical difference in the horizontal ladder walking was found between the groups, the increase in collateral sprouting and in the number of contacts correlated with the functional recovery. Hence, cell body treatment can promote plasticity of the injured CNS and may be a valuable treatment approach in conjunction with local regeneration promoting strategies.

Metabolism-resistant bradykinin antagonists: development and applications.

Bradykinin plays many roles in normal and pathological physiology, but rapid enzymatic degradation made elucidation of its functions extremely difficult. Development of effective degradation-resistant antagonists made it possible to delineate these roles and to open the way for development of new drugs to control pathology due to excess production of bradykinin. Presently available peptide bradykinin antagonists are extremely potent, are completely resistant to enzymatic degradation, and are orally available. Non-peptide bradykinin antagonists have also been discovered. Development of bradykinin antagonists as drugs for cancer, inflammation and trauma is anticipated.

Characterization of receptor-mediated actions of T-kinin.

T-Kinin (Ile-Ser-bradykinin) is unique to the rat. This study characterizes the receptors involved in T-kinin activity on both the intact isolated rat uterus and membrane receptor preparations of the rat uterus. The results show that T-kinin acts through kinin B2 receptors in the rat uterus as demonstrated by B2 receptor-antagonist inhibition. While the potency of T-kinin on rat uterus contraction was similar to that of bradykinin, binding studies showed that the affinity of T-kinin to the receptor was 10-fold lower than that of bradykinin. On the other hand, the D isomer of T-kinin, D-Ile-Ser-bradykinin, had an affinity for the receptor greater than that of T-kinin and was more potent in causing contraction. Comparing this finding with our previously published report that D-Ile-Ser-bradykinin is not active on the kinin receptor for vascular permeability indicates that the kinin receptors in the rat uterus are not the same as those previously reported in the smooth muscle of the vasculature, i.e. there exists subclasses of kinin B2 receptors. The data from binding studies on a variety of T-kinin analogues show that the substitution of hydroxyproline (Hyp) for Pro5, together with the D-configuration at Ile1 and/or Ser2 may be useful for the development of selective T-kinin antagonists. Studies involving pretreatment of the tissue with indomethacin demonstrated that prostaglandin release was more of a component of T-kinin's activity on the rat uterus than that of bradykinin.

Diversity of B2 bradykinin receptors with nanomolar affinity expressed in passaged IMR90 human lung fibroblasts.

IMR90 human fetal lung fibroblasts express bradykinin receptors activating the pathway for biosynthesis of PGE2. A receptor of the B2 subtype stimulates half-maximal PGE2 production at 4.8 nM bradykinin, and maximal output takes place at 25 nM bradykinin. Radioligand binding studies reveal a population of [3H]bradykinin binding sites whose affinity correlates with this B2 receptor's biologic activity, with a KD of 2.5 nM. As IMR90 cells reach 60% of their defined life span in culture, they spontaneously induce expression of a second site of lower affinity, with half-maximal binding of [3H]bradykinin at 44 nM. This second site displays a characteristic primary B2 receptor recognition profile, but differs from the 2.5 nM site on a secondary level in recognition among different B2 ligands. Bradykinin is the most potent ligand at both sites; they each preferentially recognize an N-terminal extended bradykinin peptide construct having selectivity for the rat myometrial B2 receptor, suggesting that both sites have structural features in common. However, they display diversity in their order of preference for Met-Lys-bradykinin versus Lys-Lys-bradykinin; at the 44 nM site this order is completely reversed from the order of potency exhibited at the 2.5 nM site. Expression of the second site changes the manner in which these fibroblasts control their PGE2 production; it affords a graded response of PGE2 production at bradykinin levels beyond those which would normally saturate the 2.5 nM site. The inducibility of the 44 nM site in cultured fibroblasts addresses in vivo conditions in an inflammatory environment where continuing generation of bradykinin-related peptides takes place and presents a possible mechanism for overriding constraints that would otherwise limit the progression of inflammation.

Evidence for renal kinins as mediators of amino acid-induced hyperperfusion and hyperfiltration in the rat.

This study examined the role of tissue kallikrein and kinins in renal vasodilation produced by infusion of amino acids (AA). In rats fed a 9% protein diet for 2 wk, intravenous infusion of a 10% AA solution over 60-90 min reduced total renal vascular resistance and increased glomerular filtration rate (GFR) by 25-40% and renal plasma flow (RPF) by 23-30% from baseline. This was associated with a two- to threefold increase in urinary kinin excretion rate. Acute treatment of rats with aprotinin, a kallikrein inhibitor, resulted in deposition of immunoreactive aprotinin in kallikrein-containing connecting tubule cells and inhibited renal kallikrein activity by 90%. A protinin pretreatment abolished the rise in urinary kinins and prevented significant increases in GFR and RPF in response to AA. In a second group of rats pretreated with a B2 kinin receptor antagonist, [DArg Hyp3, Thi5,8 D Phe7]bradykinin, AA infusion raised urinary kinins identically as in untreated controls, but GFR and RPF responses were absent. Aprotinin or the kinin antagonist produced no consistent change in renal function in rats that were not infused with AA.AA-induced increases in kinins were not associated with an increase in renal kallikrein activity. Notably, tissue active kallikrein level fell 50% in AA-infused rats. These studies provide evidence that kinins generated in the kidney participate in mediating renal vasodilation during acute infusion of AA.

Influence of a kinin antagonist on acute hypotensive responses induced by bradykinin and captopril in spontaneously hypertensive rats.

We have evaluated the effects of a B2 receptor antagonist (B5630) of kinins on BK and captopril-induced acute hypotensive responses in anaesthetized SHR. Intravenous treatment of BK (1.0 microgram) and captopril (0.3 mg/kg) caused significant (p < 0.05) fall in the SBP and DBP. Whereas BK caused greater fall in the SBP (p < 0.05), DBP (p < 0.01) and duration of hypotension (p < 0.05) when administered after captopril (Fig 1 and 2). All the hypotensive effects of BK and captopril were significantly antagonised (p < 0.05) in the presence of B5630 (2.0 mg/kg). Further, the duration of hypotensive responses of BK and captopril were blocked (p < 0.05) by B5630. The agonists and BK-antagonist did not cause significant (p > 0.05) alterations in HR during the entire investigation. These findings provide evidence to support the suggestion that B2 receptor might be involved in the regulation of the hypotensive actions of BK and captopril. Kinins should also have valuable functions in the antihypertensive property of captopril-like drugs.

Pseudopeptide analogs of bradykinin and bradykinin antagonists.

Over 100 reduced-bond analogs of bradykinin and bradykinin antagonists were designed, synthesized, and assayed in the classic smooth muscle and blood pressure assays in a further study of the structure-activity relationships of bradykinin. Both potent BK-like agonists and antagonists of BK activity were found among the reduced-bond analogs.

Bradykinin antagonists do not require a D-aromatic amino acid residue at position 7.

More than 200 new analogs of bradykinin have been synthesized and assayed. Analogs containing a D-aliphatic amino acid residue at position 7 and an L-aliphatic residue at position 8 are very potent antagonists of BK action on classical systems. Thus the "conventional wisdom" that a D-aromatic amino acid residue is required at sequence position 7 of BK antagonists must be modified.

Evaluation of cutaneous algogenic responses to bradykinin and inhibition by bradykinin analogs.

Bradykinin has long been postulated to have a major role in physiologic human pain production. We have developed a method to systematically quantify the algogenic response to bradykinin in human skin by the direct application of bradykinin to suction blister bases. After determination of the pain threshold to bradykinin, we examined the ability of synthetic peptide analogs of bradykinin to block bradykinin-induced pain. One of the six analogs (B4642) inhibited bradykinin-induced pain with at least one concentration in all subjects tested. This result could not be predicted on the basis of inhibition of bradykinin binding to its guinea-pig ileum receptor or to previous studies of bradykinin-induced pain production in animal models. This peptide may have an eventual role as a physiologic pain inhibitor.

Bradykinin-induced burst of prostaglandin formation in osteoblasts is mediated via B2 bradykinin receptors.

The receptor subtype mediating the bradykinin (Bk)-induced burst of prostaglandin formation in osteoblasts has been studied. Bk, but not des-Arg9-Bk, induced the formation of prostaglandin E2 and prostacyclin in neonatal mouse calvarial bones incubated for 30 minutes. Bk-induced prostaglandin synthesis is neonatal mouse calvarial bones was significantly inhibited by the B2 Bk receptor antagonist D-Arg0[Hyp3,Thi5,8,D-Phe7]-Bk. The B2 Bk receptor agonists Bk and Lys-Bk, but not the B1 Bk receptor agonist des-Arg9-Bk, caused a rapid burst (5 minutes) of prostaglandin E2 and prostacyclin formation in isolated osteoblast-like cells from neonatal mouse calvarial bones and in the murine osteoblastic cell lineage MC3T3-E1. When comparing the relative potency of different kinin analogs on their stimulatory effect on prostaglandin formation in isolated osteoblast-like cells and in MC3T3-E1 cells, we found that Bk = Lys-Bk greater than Met-lys-Bk much much greater than Des-Arg9-Bk. Bk-induced prostaglandin synthesis in isolated osteoblast-like cells and in MC3T3-E1 cells was inhibited by D-Arg0[Hyp3,Thi5,8,D-Phe7]-Bk, whereas the B1 Bk receptor antagonist des-Arg9-Leu8-Bk had no effect. Total binding of 3H-Bk (3-20 nM; 4 degrees C) to whole MC3T3-E1 cells reached a maximum after 4-5 h. An excess of nonradioactive Bk (1 microM) reduced cell-associated radioactivity by 20-30%. The B2 Bk receptor agonist Bk and the B2 Bk receptor antagonist D-Arg0[Hyp3,Thi5,8,D-Phe7]-Bk were able to reduce specific binding, but the B1 Bk receptor agonist des-Arg9-Bk was unable to reduce the specific binding of 3H-Bk to whole MC3T3-E1 cells. These findings indicate the presence of B2 Bk receptors on osteoblasts coupled to enhanced prostaglandin synthesis.

Bradykinin and its Gly6 analogue are substrates of cyclophilin: a fluorine-19 magnetization transfer study.

Fluorine-19 magnetization transfer experiments have been used to determine the rates of cis/trans isomerization about the X-Pro7 peptide bond in [p-fluoro-Phe8]bradykinin (cis/trans ratio approximately 0.1) and its Gly6 analogue (cis/trans ratio approximately 0.4). The measurements were carried out both prior to and after the addition of cyclophilin, which has recently been shown to have peptidyl-proline cis/trans isomerase activity and is the apparent target enzyme of the immunosuppressive agent cyclosporin A. Magnetization transfer measurements over the temperature range 40-75 degrees C in the absence of enzyme give activation energies of 22.8 and 23.0 kcal/mol for [p-fluoro-Phe8]bradykinin and its Gly6 analogue, respectively. The values for the uncatalyzed cis----trans rate constant, kc, are determined by extrapolation to be 4.8 x 10(-2) and 2.1 x 10(-2) s-1 for the two peptides at 25 degrees C. The enzyme-catalyzed enhancement of the cis/trans interconversion rate was proportional to added cyclophilin concentration and was strongly sequence specific, with bradykinin a much better substrate than [Gly6]bradykinin. At a peptide concentration of 2.2 mM, the catalytic activity expressed as kc per micromolar cyclophilin was determined to be 1.2 s-1/microM for [p-fluoro-Phe8]bradykinin and 0.13 s-1/microM for the Gly6 analogue. The increased cis----trans interconversion rates were strongly inhibited by cyclosporin A and the 6-(methylalanine) derivative, which bind to cyclophilin, but not by the 1-(tetrahydrofurfuryl) derivative of cyclosporin that binds weakly.

Echo-planar imaging of intravoxel incoherent motion.

The recently established single-shot technique of echo-planar imaging of intravoxel incoherent motion (IVIM) for determining and imaging the variations of microscopic motions of water has been applied to studies of water perfusion in phantoms and to in vivo studies of diffusion and perfusion in cat and human brains. The phantom results demonstrate that perfusion levels comparable with those found in vivo have easily observable and reproducible effects on signal amplitude that are consistent with previous IVIM theory. Reliable measurements of the diffusion coefficient in various types of brain tissue have been obtained. The results for white matter are consistent with the existence of anisotropic diffusion in oriented bundles of myelinated nerve fibers. The results for gray matter can be fitted to the IVIM theory and suggest a value of up to 14% for the fraction of the signal contributed by randomly perfusing fluid in normal cerebral cortex.

Luminal responses to bradykinin on the isolated canine tracheal epithelium: effects of bradykinin antagonists.

We have tested the ability of several B2 antagonists on the responses of the open-circuited isolated canine tracheal epithelium to the luminal addition of Bradykinin (BK), Lys-BK, and substance P (SP). All three peptides produced biphasic changes in transmural potential difference (PD), an initial decrease (dip) followed by an increase (rise). The B2 antagonists D-Arg0 [Hyp3,Thi5,8,D-Phe7]BK (B5630) reversibly inhibited both the dips and the rise with IC50 values of 2.01 x 10(-8) and 1.54 x 10(-7) M, respectively. The responses to SP were unaffected even with high concentrations of the antagonist. Other antagonists tested [D-Phe1,7,Thi5,8]BK (B4158), [D-Phe2,7]BK (B4404), and [D-Phe7,Hyp8]BK (B5092) were ineffective.

Effects of a kinin antagonist on renal function in rats.

We contrasted the effects of D-Arg-Arg-Pro-Hyp-Gly-Thi-Ser-DPhe-Thi-Arg-TFA (kinin receptor antagonist), of aprotinin (kallikrein inhibitor), and of combined treatment with captopril (kininase II inhibitor) and phosphoramidon (neutral endopeptidase 24.11 inhibitor) on renal function of rats with and without 14-day deoxycorticosterone pretreatment (DOC, 25 mg.kg-1.wk-1 sc). Neither the kinin antagonist nor aprotinin affected renal function in rats with and without DOC pretreatment. Combined treatment with captopril and phosphoramidon caused in rats with and without DOC pretreatment augmentation (P less than 0.05) of kinin excretion (50-64%), glomerular filtration rate (12-11%), and sodium excretion (46-48%). In DOC-pretreated rats undergoing infusion of captopril and phosphoramidon, the superimposed administration of either the kinin antagonist or aprotinin caused the lowering of renal plasma flow, glomerular filtration rate, and sodium excretion. These effects of the kinin antagonist and aprotinin in rats infused with kininase inhibitors may be the consequence of blockade, respectively, of the renal actions and synthesis of kinins that, when in excess, elicit renal vasodilation and increase glomerular filtration rate and sodium excretion. Collectively, these observations suggest regulatory influence of kinins during conditions featuring increased renal kinin levels.

Effects of kinins on cultured arterial smooth muscle.

The present study uses various kinin agonists and antagonists to examine the cellular mechanisms of bradykinin's actions on intracellular calcium, prostaglandins, and adenosine 3',5'-cyclic monophosphate (cAMP) accumulation in cultured arterial smooth muscle cells (casmc) obtained from rat mesenteric arteries. Exposure to bradykinin produced a rapid release of calcium (peak less than or equal to 20 s) from intracellular stores and an increase in prostaglandin (PG) E2 and cAMP production in casmc. Compared with bradykinin, the bradykinin B1-agonist [des-Arg9]BK produced only a small increase in intracellular calcium. The bradykinin-mediated increase in intracellular calcium was competitively blocked by the B2 receptor antagonist [D-Arg-O-Hyp3-Thi5,8-D-Phe7]BK (B4307) but not the B1-antagonist ([des-Arg9-Leu8]BK). In addition, the similarity of the dose-response curves for the bradykinin-mediated increase in Ca2+, PGE2, and cAMP (half-maximal stimulation of 12, 11, and 13 nM, respectively) and the ability of the B2-antagonist (B4307) to block each of these effects of bradykinin suggest that all three effects are mediated by the same bradykinin (B2) receptor. Further studies revealed that increases in intracellular calcium are necessary for the bradykinin-mediated increase in PGE2 formation and the subsequent PGE2-dependent formation of cAMP. Taken together, these results suggest that bradykinin acts via a B2-receptor on arterial smooth muscle cells to release calcium from intracellular stores, leading to increases in PGE2 production and the PGE2-dependent activation of adenylate cyclase.