Understanding the mechanism of polyethyleneimine-mediated cell disintegration and protein extraction in E. coli: The role of floc network formation and PEI molecular weight.

Cell disintegration and protein extraction are crucial steps in downstream process development for biopharmaceuticals produced in E. coli. In this study, we explored the extraction mechanism of polyethyleneimine (PEI) at the cellular level and characterized the floc network that is formed upon PEI addition by Focused Beam Reflectance Measurement and Dispersion Analyzer. PEI disintegrates the cells by detachment of the outer membrane allowing protein to diffuse into the interspace of the flocs. Protein release into the supernatant occurs by diffusion out of the floc network. We could show that the type and concentrations of PEIs with varying molecular weight determines the floc properties and thus the extraction efficiency. We could demonstrate why optimal conditions, using 70 kDa PEI at 0.25 g/g cell dry mass, lead to efficient extraction while at suboptimal conditions extraction is almost negligible. Our findings provide valuable insights into the relationship between floc properties and PEI-driven protein extraction, with potential applications in bioprocessing and biotechnology.

Knockdown of calpain1 in lumbar motoneurons reduces spasticity after spinal cord injury in adult rats.

Spasticity, affecting ∼75% of patients with spinal cord injury (SCI), leads to hyperreflexia, muscle spasms, and cocontractions of antagonist muscles, greatly affecting their quality of life. Spasticity primarily stems from the hyperexcitability of motoneurons below the lesion, driven by an upregulation of the persistent sodium current and a downregulation of chloride extrusion. This imbalance results from the post-SCI activation of calpain1, which cleaves Nav1.6 channels and KCC2 cotransporters. Our study was focused on mitigating spasticity by specifically targeting calpain1 in spinal motoneurons. We successfully transduced lumbar motoneurons in adult rats with SCI using intrathecal administration of adeno-associated virus vector serotype 6, carrying a shRNA sequence against calpain1. This approach significantly reduced calpain1 expression in transduced motoneurons, leading to a noticeable decrease in spasticity symptoms, including hyperreflexia, muscle spasms, and cocontractions in hindlimb muscles, which are particularly evident in the second month post-SCI. In addition, this decrease, which prevented the escalation of spasticity to a severe grade, paralleled the restoration of KCC2 levels in transduced motoneurons, suggesting a reduced proteolytic activity of calpain1. These findings demonstrate that inhibiting calpain1 in motoneurons is a promising strategy for alleviating spasticity in SCI patients.

Persistent Nav1.1 and Nav1.6 currents drive spinal locomotor functions through nonlinear dynamics.

Persistent sodium current (INaP) in the spinal locomotor network promotes two distinct nonlinear firing patterns: a self-sustained spiking triggered by a brief excitation in bistable motoneurons and bursting oscillations in interneurons of the central pattern generator (CPG). Here, we identify the NaV channels responsible for INaP and their role in motor behaviors. We report the axonal Nav1.6 as the main molecular player for INaP in lumbar motoneurons. The inhibition of Nav1.6, but not of Nav1.1, in motoneurons impairs INaP, bistability, postural tone, and locomotor performance. In interneurons of the rhythmogenic CPG region, both Nav1.6 and Nav1.1 equally mediate INaP. Inhibition of both channels is required to abolish oscillatory bursting activities and the locomotor rhythm. Overall, Nav1.6 plays a significant role both in posture and locomotion by governing INaP-dependent bistability in motoneurons and working in tandem with Nav1.1 to provide INaP-dependent rhythmogenic properties of the CPG.

L-Arginine sulfate reduces irreversible protein binding in immobilized metal affinity chromatography.

Immobilized metal affinity chromatography (IMAC) is a powerful technique for capture and purification of relevant biopharmaceuticals in complex biological matrices. However, protein recovery can be drastically compromised due to surface induced spreading and unfolding of the analyte, leading to fouling of the stationary phase. Here, we report on the kinetics of irreversible adsorption of a protease on an IMAC resin in a time span ranging from minutes to several hours. This trend correlated with the thermal data measured by nano differential scanning calorimetry, and showed a time-dependent change in protein unfolding temperature. Our results highlight that 'soft' proteins show a strong time dependent increase in irreversible adsorption. Furthermore, commonly used co-solvents for preservation of the native protein conformation are tested for their ability to reduce fouling. Thermal data suggests that the amino acid l-arginine is beneficial in preventing unfolding, which was confirmed in batch adsorption experiments. The choice of counter-ions has to be considered when using this amino acid. These results show that l-arginine sulfate decelerates the irreversible adsorption kinetics of proteins on the IMAC stationary phase to a greater extent than l-arginine chloride.

Polyethyleneimine efficiently extracts recombinant cytoplasmatic green fluorescent protein produced in Escherichia coli with high purity.

We used a polycationic polymer polyethyleneimine (PEI) to develop a method to extract recombinant proteins produced in the Escherichia coli (E. coli) cytosol. Compared to high pressure homogenization, commonly used to disrupt E. coli cells, our extraction method leads to higher purity of extracts. Upon addition of PEI to the cells, flocculation occurs and the recombinant protein gradually diffuses out of the PEI/cell network. While several aspects such as the E. coli strain, the cell or PEI concentration as well as the protein titer and the pH of the buffer seem to influence the extraction rate, our results show that the PEI molecule (molecular weight and structure) must be chosen appropriately for protein extraction. The method works well with resuspended cells but can also be applied directly to fermentation broths at higher PEI concentration. This extraction approach allows for effective reduction of DNA, endotoxins, and host cell proteins levels by 2-4 orders of magnitude, and drastically facilitate the subsequent downstream processing steps such as centrifugation and filtration.

Riluzole for treating spasticity in patients with chronic traumatic spinal cord injury: Study protocol in the phase ib/iib adaptive multicenter randomized controlled RILUSCI trial.

BACKGROUND: Satisfactory treatment is often lacking for spasticity, a highly prevalent motor disorder in patients with spinal cord injury (SCI). Low concentrations of riluzole potently reduce the persistent sodium current, the post-SCI increase in which contributes to spasticity. The repurposing of this drug may therefore constitute a useful potential therapeutic option for relieving SCI patients suffering from chronic traumatic spasticity. OBJECTIVE: RILUSCI is a phase 1b-2b trial designed to assess whether riluzole is a safe and biologically effective means of managing spasticity in adult patients with traumatic chronic SCI. METHODS: In this multicenter double-blind trial, adults (aged 18-65 years) suffering from spasticity after SCI (target enrollment: 90 participants) will be randomly assigned to be given either a placebo or a recommended daily oral dose of riluzole for two weeks. The latter dose will be previously determined in phase 1b of the study by performing double-blind dose-finding tests using a Bayesian continuous reassessment method. The primary endpoint of the trial will be an improvement in the Modified Ashworth Score (MAS) or the Numerical Rating Score (NRS) quantifying spasticity. The secondary outcomes will be based on the safety and pharmacokinetics of riluzole as well as its impact on muscle spasms, pain, bladder dysfunction and quality of life. Analyses will be performed before, during and after the treatment and the placebo-controlled period. CONCLUSION: To the best of our knowledge, this clinical trial will be the first to document the safety and efficacy of riluzole as a means of reducing spasticity in patients with chronic SCI. TRIAL REGISTRATION: The clinical trial, which is already in progress, was registered on the ClinicalTrials.gov website on August 9, 2016 under the registration number NCT02859792. TRIAL SPONSOR: Assistance Publique-Hôpitaux de Marseille.

Refolding in the modern biopharmaceutical industry.

Inclusion bodies (IBs) often emerge upon overexpression of recombinant proteins in E. coli. From IBs, refolding is necessary to generate the native protein that can be further purified to obtain pure and active biologicals. This work focusses on refolding as a significant process step during biopharmaceutical manufacturing with an industrial perspective. A theoretical and historical background on protein refolding gives the reader a starting point for further insights into industrial process development. Quality requirements on IBs as starting material for refolding are discussed and further economic and ecological aspects are considered with regards to buffer systems and refolding conditions. A process development roadmap shows the development of a refolding process starting from first exploratory screening rounds to scale-up and implementation in manufacturing plant. Different aspects, with a direct influence on yield, such as the selection of chemicals including pH, ionic strength, additives, etc., and other often neglected aspects, important during scale-up, such as mixing, and gas-fluid interaction, are highlighted with the use of a quality by design (QbD) approach. The benefits of simulation sciences (process simulation and computer fluid dynamics) and process analytical technology (PAT) for seamless process development are emphasized. The work concludes with an outlook on future applications of refolding and highlights open research inquiries.

CASPON platform technology: Ultrafast circularly permuted caspase-2 cleaves tagged fusion proteins before all 20 natural amino acids at the N-terminus.

Fusion protein technologies improve the expression and purification of recombinant proteins, but the removal of the tags involved requires specific proteases. The circularly permuted caspase-2 (cpCasp2) with its specific cleavage site, efficiently generates the untagged protein. While cleavage with cpCasp2 is possible before all 20 proteinogenic amino acids, cleavage before valine, leucine, isoleucine, aspartate and glutamate suffers from slow, and before proline extremely slow, turnover. To make the platform fusion protein process even more general such that any protein with an authentic N-terminus can be produced with high efficiency, the bacterial selection system PROFICS (PRotease Optimization via Fusion-Inhibited Carbamoyltransferase-based Selection) was used to evolve cpCasp2 into a variant with a catalytic turnover two orders of magnitude higher and the ability to cleave before any amino acid. The high specificity and the stability of the original circularly permuted protease was fully retained in this mutant, while the high manufacturability was mostly retained, albeit with decreased soluble titer. Four point-mutations are responsible for this change in activity, two of which are located in or near the binding pocket of the active site. This variant was named CASPON enzyme and is a major component of the CASPase-based fusiON (CASPON) platform technology. Applicability for the production of recombinant proteins was demonstrated by enzymatic removal of the CASPON tag from five model proteins. The CASPON tag enables high soluble expressions, affinity purification and good accessibility for cleavage. The five industry-relevant proteins of interest were FGF2, TNF, GH, GCSF and PTH.

Smart process development: Application of machine-learning and integrated process modeling for inclusion body purification processes.

The development of a biopharmaceutical production process usually occurs sequentially, and tedious optimization of each individual unit operation is very time-consuming. Here, the conditions established as optimal for one-step serve as input for the following step. Yet, this strategy does not consider potential interactions between a priori distant process steps and therefore cannot guarantee for optimal overall process performance. To overcome these limitations, we established a smart approach to develop and utilize integrated process models using machine learning techniques and genetic algorithms. We evaluated the application of the data-driven models to explore potential efficiency increases and compared them to a conventional development approach for one of our development products. First, we developed a data-driven integrated process model using gradient boosting machines and Gaussian processes as machine learning techniques and a genetic algorithm as recommendation engine for two downstream unit operations, namely solubilization and refolding. Through projection of the results into our large-scale facility, we predicted a twofold increase in productivity. Second, we extended the model to a three-step model by including the capture chromatography. Here, depending on the selected baseline-process chosen for comparison, we obtained between 50% and 100% increase in productivity. These data show the successful application of machine learning techniques and optimization algorithms for downstream process development. Finally, our results highlight the importance of considering integrated process models for the whole process chain, including all unit operations.

Prenatal Hypoxia Induces Cl- Cotransporters KCC2 and NKCC1 Developmental Abnormality and Disturbs the Influence of GABAA and Glycine Receptors on Fictive Breathing in a Newborn Rat.

Prenatal hypoxia is a recognised risk factor for neurodevelopmental disorders associated with both membrane proteins involved in neuron homeostasis, e.g., chloride (Cl-) cotransporters, and alterations in brain neurotransmitter systems, e.g., catecholamines, dopamine, and GABA. Our study aimed to determine whether prenatal hypoxia alters central respiratory drive by disrupting the development of Cl- cotransporters KCC2 and NKCC1. Cl- homeostasis seems critical for the strength and efficiency of inhibition mediated by GABAA and glycine receptors within the respiratory network, and we searched for alterations of GABAergic and glycinergic respiratory influences after prenatal hypoxia. We measured fictive breathing from brainstem in ex vivo preparations during pharmacological blockade of KCC2 and NKCC1 Cl- cotransporters, GABAA, and glycine receptors. We also evaluated the membrane expression of Cl- cotransporters in the brainstem by Western blot and the expression of Cl- cotransporter regulators brain-derived neurotrophic factor (BDNF) and calpain. First, pharmacological experiments showed that prenatal hypoxia altered the regulation of fictive breathing by NKCC1 and KCC2 Cl- cotransporters, GABA/GABAA, and glycin. NKCC1 inhibition decreased fictive breathing at birth in control mice while it decreased at 4 days after birth in pups exposed to prenatal hypoxia. On the other hand, inhibition of KCC2 decreased fictive breathing 4 days after birth in control mice without any change in prenatal hypoxia pups. The GABAergic system appeared to be more effective in prenatal hypoxic pups whereas the glycinergic system increased its effectiveness later. Second, we observed a decrease in the expression of the Cl- cotransporter KCC2, and a decrease with age in NKCC1, as well as an increase in the expression of BDNF and calpain after prenatal hypoxia exposure. Altogether, our data support the idea that prenatal hypoxia alters the functioning of GABAA and glycinergic systems in the respiratory network by disrupting maturation of Cl- homeostasis, thereby contributing to long-term effects by disrupting ventilation.

Early Hypoexcitability in a Subgroup of Spinal Motoneurons in Superoxide Dismutase 1 Transgenic Mice, a Model of Amyotrophic Lateral Sclerosis.

In amyotrophic lateral sclerosis (ALS), large motoneurons degenerate first, causing muscle weakness. Transgenic mouse models with a mutation in the gene encoding the enzyme superoxide dismutase 1 (SOD1) revealed that motoneurons innervating the fast-fatigable muscular fibres disconnect very early. The cause of this peripheric disconnection has not yet been established. Early pathological signs were described in motoneurons during the postnatal period of SOD1 transgenic mice. Here, we investigated whether the early changes of electrical and morphological properties previously reported in the SOD1G85R strain also occur in the SOD1G93A-low expressor line with particular attention to the different subsets of motoneurons defined by their discharge firing pattern (transient, sustained, or delayed-onset firing). Intracellular staining and recording were performed in lumbar motoneurons from entire brainstem-spinal cord preparations of SOD1G93A-low transgenic mice and their WT littermates during the second postnatal week. Our results show that SOD1G93A-low motoneurons exhibit a dendritic overbranching similar to that described previously in the SOD1G85R strain at the same age. Further we found an hypoexcitability in the delayed-onset firing SOD1G93A-low motoneurons (lower gain and higher voltage threshold). We conclude that dendritic overbranching and early hypoexcitability are common features of both low expressor SOD1 mutants (G85R and G93A-low). In the high-expressor SOD1G93A line, we found hyperexcitability in the sustained firing motoneurons at the same period, suggesting a delay in compensatory mechanisms. Overall, our results suggest that the hypoexcitability indicate an early dysfunction of the delayed-onset motoneurons and could account as early pathological signs of the disease.

Integrated process development: The key to improve Fab production in E. coli.

Bioprocess development and optimization is a challenging, costly, and time-consuming effort. In this multidisciplinary task, upstream processing (USP) and downstream processing (DSP) are conventionally considered distinct disciplines. This consideration fosters "one-way" optimization disregarding interdependencies between unit operations; thus, the full potential of the process chain cannot be achieved. Therefore, it is necessary to fully integrate USP and DSP process development to provide balanced biotechnological production processes. The aim of the present study was to investigate how different host/secretory signal/antigen binding fragment (Fab) combinations in E. coli expression systems influence USP, primary recovery performance and the final product quality. We ran identical fed-batch cultivations with 16 different expression clones to study growth and product formation kinetics, as well as centrifugation efficiency, viscosity, extracellular DNA, and endotoxin content, important parameters in DSP. We observed a severe influence on cell growth, product titer, extracellular product, and cell lysis, accompanied by a significant impact on the analyzed parameters of DSP performance. Our results provide the basis for future research on integrated process development considering interdependencies between USP and DSP; however, individual products need to be considered specifically. These interdependencies need to be understood for rational decision-making and efficient process development in research and industry.

Three-dimensional chromatography for purification and characterization of antibody fragments and related impurities from Escherichia coli crude extracts.

Antibody fragments (Fab) are often produced by recombinant methods in Escherichia coli as no glycosylation is needed. Besides the correctly expressed Fab molecule, a multitude of host cell impurities and product related impurities are present in the crude sample. The identification and characterization of the product-related impurities, such as modified Fab-molecules or free light chain, are of utmost importance. The objective of this work was to design a purification strategy to isolate and characterize Fab and related impurities. A three-dimensional chromatography method was established, consisting of two affinity steps (Protein G and Protein L) and subsequent cation exchange chromatography, followed by mass spectrometry analysis of the purified samples. The procedure was automated by collecting the eluted target species in loops and directly loading the samples onto the high-resolution cation exchange chromatography column. As an example, four different Fab molecules are characterized. All four samples contained mainly the correct Fab, while only one showed extensive N-terminal pyroglutamate formation of the Fab. In another case, we found a light chain variant with uncleaved amino acids from the lead molecule, which was not used for the formation of whole Fab as only correct Fab was found in that sample. Impurities with lower molecular weights, which were bound on the Protein L column, were observed in all samples, and identified as fragments of the light chain. In conclusion, we have devised a platform for characterizing Fab and Fab-related impurities, which significantly facilitated strain selection and optimization of cultivation conditions.

Production of Circularly Permuted Caspase-2 for Affinity Fusion-Tag Removal: Cloning, Expression in Escherichia coli, Purification, and Characterization.

Caspase-2 is the most specific protease of all caspases and therefore highly suitable as tag removal enzyme creating an authentic N-terminus of overexpressed tagged proteins of interest. The wild type human caspase-2 is a dimer of heterodimers generated by autocatalytic processing which is required for its enzymatic activity. We designed a circularly permuted caspase-2 (cpCasp2) to overcome the drawback of complex recombinant expression, purification and activation, cpCasp2 was constitutively active and expressed as a single chain protein. A 22 amino acid solubility tag and an optimized fermentation strategy realized with a model-based control algorithm further improved expression in Escherichia coli and 5.3 g/L of cpCasp2 in soluble form were obtained. The generated protease cleaved peptide and protein substrates, regardless of N-terminal amino acid with high activity and specificity. Edman degradation confirmed the correct N-terminal amino acid after tag removal, using Ubiquitin-conjugating enzyme E2 L3 as model substrate. Moreover, the generated enzyme is highly stable at -20 °C for one year and can undergo 25 freeze/thaw cycles without loss of enzyme activity. The generated cpCasp2 possesses all biophysical and biochemical properties required for efficient and economic tag removal and is ready for a platform fusion protein process.

The M-current works in tandem with the persistent sodium current to set the speed of locomotion.

The central pattern generator (CPG) for locomotion is a set of pacemaker neurons endowed with inherent bursting driven by the persistent sodium current (INaP). How they proceed to regulate the locomotor rhythm remained unknown. Here, in neonatal rodents, we identified a persistent potassium current critical in regulating pacemakers and locomotion speed. This current recapitulates features of the M-current (IM): a subthreshold noninactivating outward current blocked by 10,10-bis(4-pyridinylmethyl)-9(10H)-anthracenone dihydrochloride (XE991) and enhanced by N-(2-chloro-5-pyrimidinyl)-3,4-difluorobenzamide (ICA73). Immunostaining and mutant mice highlight an important role of Kv7.2-containing channels in mediating IM. Pharmacological modulation of IM regulates the emergence and the frequency regime of both pacemaker and CPG activities and controls the speed of locomotion. Computational models captured these results and showed how an interplay between IM and INaP endows the locomotor CPG with rhythmogenic properties. Overall, this study provides fundamental insights into how IM and INaP work in tandem to set the speed of locomotion.

Extraction of recombinant periplasmic proteins under industrially relevant process conditions: Selectivity and yield strongly depend on protein titer and methodology.

In this work, we attempted to identify a method for the selective extraction of periplasmic endogenously expressed proteins, which is applicable at an industrial scale. For this purpose, we used an expression model that allows coexpression of two fluorescent proteins, each of which is specifically targeted to either the cytoplasm or periplasm. We assessed a number of scalable lysis methods (high-pressure homogenization, osmotic shock procedures, extraction with ethylenediaminetetraacetic acid, and extraction with deoxycholate) for the ability to selectively extract periplasmic proteins rather than cytoplasmic proteins. Our main conclusion was that although we identified industrially scalable lysis conditions that significantly increased the starting purity for further purification, none of the tested conditions were selective for periplasmic protein over cytoplasmic protein. Furthermore, we demonstrated that efficient extraction of the expressed recombinant proteins was largely dependent on the overall protein concentration in the cell.

Calpain fosters the hyperexcitability of motoneurons after spinal cord injury and leads to spasticity.

Up-regulation of the persistent sodium current (INaP) and down-regulation of the potassium/chloride extruder KCC2 lead to spasticity after spinal cord injury (SCI). We here identified calpain as the driver of the up- and down-regulation of INaP and KCC2, respectively, in neonatal rat lumbar motoneurons. Few days after SCI, neonatal rats developed behavioral signs of spasticity with the emergence of both hyperreflexia and abnormal involuntary muscle contractions on hindlimbs. At the same time, in vitro isolated lumbar spinal cords became hyperreflexive and displayed numerous spontaneous motor outputs. Calpain-I expression paralleled with a proteolysis of voltage-gated sodium (Nav) channels and KCC2. Acute inhibition of calpains reduced this proteolysis, restored the motoneuronal expression of Nav and KCC2, normalized INaP and KCC2 function, and curtailed spasticity. In sum, by up- and down-regulating INaP and KCC2, the calpain-mediated proteolysis of Nav and KCC2 drives the hyperexcitability of motoneurons which leads to spasticity after SCI.

Mild Intrauterine Hypoperfusion Leads to Lumbar and Cortical Hyperexcitability, Spasticity, and Muscle Dysfunctions in Rats: Implications for Prematurity.

Intrauterine ischemia-hypoxia is detrimental to the developing brain and leads to white matter injury (WMI), encephalopathy of prematurity (EP), and often to cerebral palsy (CP), but the related pathophysiological mechanisms remain unclear. In prior studies, we used mild intrauterine hypoperfusion (MIUH) in rats to successfully reproduce the diversity of clinical signs of EP, and some CP symptoms. Briefly, MIUH led to inflammatory processes, diffuse gray and WMI, minor locomotor deficits, musculoskeletal pathologies, neuroanatomical and functional disorganization of the primary somatosensory and motor cortices, delayed sensorimotor reflexes, spontaneous hyperactivity, deficits in sensory information processing, memory and learning impairments. In the present study, we investigated the early and long-lasting mechanisms of pathophysiology that may be responsible for the various symptoms induced by MIUH. We found early hyperreflexia, spasticity and reduced expression of KCC2 (a chloride cotransporter that regulates chloride homeostasis and cell excitability). Adult MIUH rats exhibited changes in muscle contractile properties and phenotype, enduring hyperreflexia and spasticity, as well as hyperexcitability in the sensorimotor cortex. Taken together, these results show that reduced expression of KCC2, lumbar hyperreflexia, spasticity, altered properties of the soleus muscle, as well as cortical hyperexcitability may likely interplay into a self-perpetuating cycle, leading to the emergence, and persistence of neurodevelopmental disorders (NDD) in EP and CP, such as sensorimotor impairments, and probably hyperactivity, attention, and learning disorders.

Kv1.2 Channels Promote Nonlinear Spiking Motoneurons for Powering Up Locomotion.

Spinal motoneurons are endowed with nonlinear spiking behaviors manifested by a spike acceleration whose functional significance remains uncertain. Here, we show in rodent lumbar motoneurons that these nonlinear spiking properties do not rely only on activation of dendritic nifedipine-sensitive L-type Ca2+ channels, as assumed for decades, but also on the slow inactivation of a nifedipine-sensitive K+ current mediated by Kv1.2 channels that are highly expressed in axon initial segments. Specifically, the pharmacological and computational inhibition of Kv1.2 channels occluded the spike acceleration of rhythmically active motoneurons and the correlated slow buildup of rhythmic motor output recorded at the onset of locomotor-like activity. This study demonstrates that slow inactivation of Kv1.2 channels provides a potent gain control mechanism in mammalian spinal motoneurons and has a behavioral role in enhancing locomotor drive during the transition from immobility to steady-state locomotion.

Activation of 5-HT2A Receptors Restores KCC2 Function and Reduces Neuropathic Pain after Spinal Cord Injury.

Downregulation of the potassium chloride cotransporter type 2 (KCC2) after a spinal cord injury (SCI) disinhibits motoneurons and dorsal horn interneurons causing spasticity and neuropathic pain, respectively. We showed recently (Bos et al., 2013) that specific activation of 5-HT2A receptors by TCB-2 [(4-bromo-3,6-dimethoxybenzocyclobuten-1-yl)methylamine hydrobromide] upregulates KCC2 function, restores motoneuronal inhibition and reduces SCI-induced spasticity. Here, we tested the potential analgesic effect of TCB-2 on central (thoracic hemisection) and peripheral [spared nerve injury (SNI)] neuropathic pain. We found mechanical and thermal hyperalgesia reduced by an acute administration of TCB-2 in rats with SCI. This analgesic effect was associated with an increase in dorsal horn membrane KCC2 expression and was prevented by pharmacological blockade of KCC2 with an intrathecal injection of DIOA [(dihydroindenyl)oxy]alkanoic acid]. In contrast, the SNI-induced neuropathic pain was not attenuated by TCB-2 although there was a slight increase of membrane KCC2 expression in the dorsal horn ipsilateral to the lesion. Up-regulation of KCC2 function by targeting 5-HT2A receptors, therefore, has therapeutic potential in the treatment of neuropathic pain induced by SCI but not by SNI.