Immediate early genes (IEGs) constitute a large family of genes well-known as early regulators of cell growth, differentiation signals, learning, and memory (28, 30, 36–38). Most recently, some IEGs were found also to be modulated post-trauma in sublesional spinal cord areas (27). For instance, Landry and colleagues have reported in low-thoracic spinal cord-transected mice that c-fos and nor-1 were respectively increased and decreased within a few days in the segments L1–L2, specifically in the dorsal horn and intermediate zone areas (39). In cervical or thoracic spinal cord-transected rats, increased fos-immunoreactive levels were also found as early as at 2.5 h post-trauma in lumbar laminae I–IV, VII, VIII, and X. Changes in the lumbar spinal cord of rostrally transected animals is of special interest since some of these segments (e.g., L1–L2 in mice) were shown to contain critical central pattern generator (CPG) elements (40). Given that IEGs are better known for their role in CNS development and plasticity, spontaneous changes of IEG expression (i.e., specifically c-fos and nor-1) in L1–L2 segments may be considered as among the first sublesional cellular events associated with altered cellular functions and properties post-SCI. This said, some of these changes may be associated also with other phenomena than plasticity or reorganization of spinal motor and locomotor networks. For instance, c-fos and nor-1 were used as markers in experimental models of pain and transient global ischemia suggesting a role in several functions (36, 38). In other studies, c-fos transcripts were found to drastically increase for up to 6 h near the site of lesion and to augment later in some distally located neurons (28). Such a regional- and time-dependent increase in IEG expression, which was found also in this study (39), points toward distinct roles for IEGs in cell death at the injury site versus in reorganization and plasticity at a distance from the epicenter.

Numerous regulatory changes are likely to be subsequently activated after altered IEG expression. These may include Tumor Necrosis Factor-alpha (TNF-alpha), preprodynorphin, and nitric oxide synthase (NOS), C1qb, Galectin-3 and p22(phox) which expression levels were found to increase in lumbar spinal cord segments for several days soon after a low-thoracic transection or injury in rats (29).

Other key elements including transmembranal receptors may be considered as good candidates for plasticity and reorganization of motor and locomotor networks located sublesionally following a spinal cord-transection (and probably to some extent also after partial injuries). For instance, glycine receptors and GAD-67 (enzyme in GABA synthesis) were found displaying increased expression levels in lumbar spinal cord segments 3 months after a low-thoracic transection (32, 41). Increased 5-HT_1A/7 receptor expression was detected using autoradiography with (³H) 8-OH-DPAT in lumbar spinal cord segments of spinal cord-transected cats (42). In spinal cord-transected mice, we found using in situ hybridization increased 5-HT_1A mRNA levels in L1–L2 segments in 5-HT₇-deficient mice compared with wild-types (43). This was interpreted as evidence suggesting that even greater changes may occur post-trauma in the absence of functionally closely-related genes. Results in mice revealed also increased 5-HT_2A mRNA levels in lumbar segments (laminae VII, VIII, and IX) several days after a low-thoracic transaction (22).

Although not specifically examined in spinal cord-transected mice, factors well-recognized for their role in development and plasticity have also been found to be modulated post-transection. Indeed, NGF, BDNF, and NT-3 were found displaying up-regulated levels of expression in sublesional areas of low-thoracic-transected rats (32). In contrast, BDNF and NT-3 as well as synapsin-1 were found to display lower levels of expression in the lumbar cord of low-thoracic-transected rats that were also deafferented which may suggest that these decreasing levels were associated specifically with low levels of muscle reflex activity due to deafferentation (22, 32).

We found that the body weight of this animal model significantly decreases soon after transection. A rapid loss typically ranging between 20 and 25% of initial body weight was found after only 1 week post-surgery (21). No significant return to normal levels was reported after 4 weeks although a moderate augmentation of approximately 1 g/week was found. Post-mortem measurements of individual limb masses revealed greater losses in hindlimbs (−28%) than in forelimbs (−21%). These relatively rapid adaptive changes were somewhat comparable to those generally reported soon after trauma in patients. Reasons underlying this initial weight loss are not fully understood but may involve reduced physical activity (or complete immobility), metabolic changes and hormonal deregulation. In turn, after a few years, this initial weight loss is often transformed into a weight gain leading to overweight and obesity problems in patients.

As mentioned above, hindlimbs are specifically affected in spinal cord-transected paraplegic animals. Results from our laboratory showed that part of this weight loss is due to a specific decrease in muscle mass. For instance, measurements from soleus muscles (ankle extensor) revealed a 32% decrease in mass at 1 week post-transection in mice (21). This decrease in mass was found to correspond to a proportional decrease in strength (absolute maximal tetanic force). Interestingly, in 2-month-spinal cord-transected mice, slow-twitch fibers (type I) from the soleus were found to progressively acquire some of the biochemical and contractile properties of fast-twitch fibers (fiber-type conversion). Again, evidence of comparable changes has been found in chronic SCI patients (2–4, 8).

SCI is also associated with a rapidly increasing risk of fracture in patients. In fact, nearly all SCI individuals experience a significant loss of bone mineral tissue (up to 30% in the femora) within only a few months (7, 10). Comparable changes are found in spinal cord-transected mice, where histomorphometric data from our laboratory revealed a drastic decrease in trabecular bone volume (−22%), thickness (−11%), and number (−15%) within only 1 month post-surgery (44).

After 2 months, densitometric measurements from mouse femoral bones using dual-energy X-ray absorptiometry reported a significant decrease (approximately 15% lower) of bone mineral density (BMD) and bone mineral content (BMC). There is evidence from mice suggesting that rapid bone loss in such conditions may involve a sharp decrease of osteoblastic activity and a rapid increase of osteoclastic activity (i.e., low osteocalcin and high acid phosphatase levels) (45).

Soon after SCI, there is a well-known risk of developing deep venous thrombosis (DVT) in the lower limbs and, hence, cardiovascular and pulmonary complications in patients (1). However, the specific mechanism underlying DVT formation following SCI remains poorly understood. Our model may be used to study this specific problem. We found using in vivo confocal microscopy that deep vein morphology is largely changed as rapidly as after 1 week post-spinal cord-transection (46). The femoral and saphenous veins were clearly found to largely increase (>1.5-fold) in diameter which may suggest that DVT formation after SCI is associated with venous stasis (reduced blood flow) due to blood vessel enlargement.

Immune deficiencies may lead to life-threatening complications after SCI. To examine possible factors that may contribute to this pathological condition, we used an automatic blood analyzer to characterize changes in red and white blood cell counts in spinal cord-transected mice. The results showed that total leukocyte numbers decreased by 35% at 1 week post-surgery and remained low subsequently (47). Specifically, lymphocyte numbers were reduced by as much as 53% at 1 week post-transection whereas monocytes and neutrophils generally remained unchanged. In turn, eosinophil counts were found to gradually decrease by 81% after 4 weeks. Analyses from bone marrow samples revealed similar changes.

Blood lipid profiles examined with clinical chemistry analyzer revealed decreased concentrations of cholesterols (−25%), triglycerides (−45%), low-density lipoproteins (LDL, −55%), high-density lipoproteins (HDL, −14%) but not platelets. Comparable data (e.g., low LDL-triglyceride levels) have been reported in acute SCI patients (47). Erythrocyte, platelet, hemoglobin, and hematocrit levels were either unchanged or moderately decreased (mild ­anemia) at least for 1 month post-transection.

Serum levels of testosterone, GH, DHEA, PTH, and insulin were studied using ELISA in control and spinal cord-transected mice. We found early transient changes in testosterone (−40% at 1 week) and GH (threefold increase) levels during the first 2 weeks post-transection (47). Other hormones undergo apparently sustained changes such as DHEA (−75%), PTH (−75%), and insulin levels (−84.5% at 1 week) which were consistently reduced throughout the time period studied (1 month post-surgery). Comparable changes have been found in humans since shortly after trauma, men were found displaying decreased testosterone levels for several weeks whereas increased GH and decreased PTH levels were reported in late chronic SCI patients (6). It remains unclear what role these hormonal changes may play in health degradation post-SCI. However, results in mice suggest that some of these changes may participate to immune deficiencies since high temporal correlations were found between specific anabolic hormones and immune cell count changes.

The many anatomic, systemic, and metabolic changes or adaptations reported here suggest that this mouse model may be useful to study SCI-related secondary complications typically found also in severely SCI patients. These detailed quantitative data measured in mice are critically important to properly assess and compare in subsequent studies the potential effects of innovative drug treatment candidates to prevent or reduce these adaptations and related health problems in chronic SCI individuals.

In order to examine pharmacological approaches aimed to re-activate properly each of these networks, corresponding quantitative assays had to be developed. For locomotor CPG reactivation, we created a novel quantitative assay called ACOS which stands for Average Combined Score (51). In brief, it allows assessing separately locomotor-like movements (LM) and non-locomotor movements (NLM) in the hindlimbs of spinal cord-transected mice. LM and NLM incidence, frequency, and amplitude are normally assessed during 4-min (video-recorded or not) prior to drug administration and after drug injection. A representative single score can be conveniently obtained for each animal by combining arithmetically the assessed values mentioned above as follows: ACOS  =  (NLM  +  (2  ×  LM))  ×  amplitude). One LM is defined as an entire step-like cycle consisting of an extension phase followed by a flexion phase occurring in both hindlimbs consecutively (i.e., bilaterally alternated or out-of-phase relation). Weight-bearing is not assessed and does not need to be expressed for LMs to be considered as such. One NLM is defined as one non-bilaterally coordinated movement (i.e., not followed by a flexion-extension on the other side). They include unilateral movements, jerks, brief sequences of fast-paw shaking (typically lasting 1–2 s/episode and counted as one NLM), twitches, and kicks. Amplitude is assessed simply by assigning one of three values; 0—if no movement is observed; 1—if the amplitude of most movements is less than half the range of motion of normal steps; 2—if the amplitude of most movements is at least more than half the range of motion of normal steps. Note that amplitude is assessed for LM and NLM indistinctively. Incidence is not measured per se but is obtained when averaging ACOS scores from all mice of a group. It corresponds to the number of mice (out of all mice tested in a group) in which NLMs or LMs were observed. Plantar foot placement and body-weight support may be reported as either present or not although they do not contribute to the equation used to obtain ACOS (i.e., in general, spinal cord-transected mice cannot be made to display weight-bearing stepping without drug or even with most drugs (for details, see Sect. 5.1)). Note that, in the equation, LM is multiplied by a factor of “2” for a very simple reason—even if the experimenter counts each LM as a one movement, an LM is a bilaterally alternating movement which as such is in fact constituted of two movements (one in each hindlimb). Consequently, LM values are multiplied by a factor of “2” whereas NLMs are not (typically unilateral movements) (34, 51).