A targeted proteomic multiplex CSF assay identifies increased malate dehydrogenase and other neurodegenerative biomarkers in individuals with Alzheimer's disease pathology.

Alzheimer's disease (AD) is the most common cause of dementia. Biomarkers are required to identify individuals in the preclinical phase, explain phenotypic diversity, measure progression and estimate prognosis. The development of assays to validate candidate biomarkers is costly and time-consuming. Targeted proteomics is an attractive means of quantifying novel proteins in cerebrospinal and other fluids, and has potential to help overcome this bottleneck in biomarker development. We used a previously validated multiplexed 10-min, targeted proteomic assay to assess 54 candidate cerebrospinal fluid (CSF) biomarkers in two independent cohorts comprising individuals with neurodegenerative dementias and healthy controls. Individuals were classified as 'AD' or 'non-AD' on the basis of their CSF T-tau and amyloid Aß1-42 profile measured using enzyme-linked immunosorbent assay; biomarkers of interest were compared using univariate and multivariate analyses. In all, 35/31 individuals in Cohort 1 and 46/36 in Cohort 2 fulfilled criteria for AD/non-AD profile CSF, respectively. After adjustment for multiple comparisons, five proteins were elevated significantly in AD CSF compared with non-AD CSF in both cohorts: malate dehydrogenase; total APOE; chitinase-3-like protein 1 (YKL-40); osteopontin and cystatin C. In an independent multivariate orthogonal projection to latent structures discriminant analysis (OPLS-DA), these proteins were also identified as major contributors to the separation between AD and non-AD in both cohorts. Independent of CSF Aß1-42 and tau, a combination of these biomarkers differentiated AD and non-AD with an area under curve (AUC)=0.88. This targeted proteomic multiple reaction monitoring (MRM)-based assay can simultaneously and rapidly measure multiple candidate CSF biomarkers. Applying this technique to AD we demonstrate differences in proteins involved in glucose metabolism and neuroinflammation that collectively have potential clinical diagnostic utility.

Mechanical evaluation of a tapered thread-run-out half-pin designed for external skeletal fixation in small animals.

An in vitro mechanical study was performed to compare the stiffness, maximum load, and cyclic load-to-failure of a new external fixation half-pin design utilizing a tapered thread-run-out (TRO) feature with currently available positive profile thread (PP) half-pins. Five different sizes of each of the two pin types were evaluated. Under static loading, TRO pins were significantly stiffer and had a higher maximum load compared to the similar sized PP pins (p <0.0001 for all comparisons). In cyclic fatigue testing, TRO pins lasted 2.3- to 4.9-fold more cycles than PP pins of similar size (p <0.0001 for all comparisons). The increased pin stiffness and improved cyclic lifespan provided by TRO pins may be especially valuable in the stabilization of biologically and mechanically challenging fracture cases where healing is prolonged.

An imposed oscillating electrical field improves the recovery of function in neurologically complete paraplegic dogs.

We show that an applied electric field in which the polarity is reversed every 15 minutes can improve the outcome from severe, acute spinal cord injury in dogs. This study utilized naturally injured, neurologically complete paraplegic dogs as a model for human spinal cord injury. The recovery of paraplegic dogs treated with oscillating electric field stimulation (OFS) (approximately 500 to 600 microV/mm; n = 20) was compared with that of sham-treated animals (n = 14). Active and sham stimulators were fabricated in West Lafayette, Indiana. They were coded, randomized, sterilized, and packaged in Warsaw, Indiana, and returned to Purdue University for blinded surgical implantation. The stimulators were of a previously unpublished design and meet the requirements for phase I human clinical testing. All dogs were treated within 18 days of the onset of paraplegia. During the experimental applications, all received the highest standard of conventional management, including surgical decompression, spinal stabilization (if required), and acute administration of methylprednisolone sodium succinate. A radiologic and neurologic examination was performed on every dog entering the study, the latter consisting of standard reflex testing, urologic tests, urodynamic testing, tests for deep and superficial pain appreciation, proprioceptive placing of the hind limbs, ambulation, and evoked potential testing. Dogs were evaluated before and after surgery and at 6 weeks and 6 months after surgery. A greater proportion of experimentally treated dogs than of sham-treated animals showed improvement in every category of functional evaluation at both the 6-week and 6-month recheck, with no reverse trend. Statistical significance was not reached in comparisons of some individual categories of functional evaluation between sham-treated and OFS-treated dogs (ambulation, proprioceptive placing); an early trend towards significance was shown in others (deep pain), and significance was reached in evaluations of superficial pain appreciation. An average of all individual scores for all categories of blinded behavioral evaluation (combined neurologic score) was used to compare group outcomes. At the 6-month recheck period, the combined neurologic score of OFS-treated dogs was significantly better than that of control dogs (p = 0.047; Mann-Whitney, two-tailed).

In vivo observations of hydraulic stiffening in the canine femoral head.

The role that intertrabecular contents and their boundary conditions have on the dynamic mechanical response of canine femoral heads was investigated in vivo. Femoral heads from paired intact hind limbs of canine specimens were subjected to a sinusoidal strain excitation at physiologic frequencies, in the cranio-caudal direction. The fluid boundary conditions for the contralateral limbs were changed by predrilling through the lateral femoral cortex and into the femoral neck. The drilling procedure did not invade the head itself. This femoral head fluid boundary alteration reduced the stiffness by 19 percent for testing at 1 Hz. The results of this study demonstrate that fluid stiffening occurs in vivo as previously observed ex vivo.

Randomized, controlled trial of the efficacy of carprofen, a nonsteroidal anti-inflammatory drug, in the treatment of osteoarthritis in dogs.

Seventy dogs were included in a randomized, controlled, multicenter trial to test the efficacy of carprofen (2.2 mg/kg of body weight, PO, q 12 h) for relief of clinical signs associated with osteoarthritis. Thirty-six dogs received carprofen, and 34 received a placebo. Response of the dogs was evaluated by comparing results of force plate examination and a graded lameness examination performed before and immediately after 2 weeks of treatment, and by obtaining a subjective assessment of the dog's posttreatment condition from owners and participating veterinarians. A physical examination, CBC, serum biochemical analyses, urinalysis, and fecal occult blood test were performed before and after treatment to monitor safety. For force plate evaluation, the odds ratio was 3.3, meaning that a dog treated with carprofen was 3.3 times more likely to have a positive response than was a dog treated with the placebo. For evaluation by a veterinarian, the odds ratio was 3.5, and for owner evaluation, the odds ratio was 4.2. Institution where dogs were treated did not have a significant effect on results. A variety of reactions that may have been related to the medication (placebo or carprofen) were recorded; however, none were considered serious. Serum alanine aminotransferase activity was high in 3 dogs (2 that received placebo and 1 that received carprofen) at the conclusion of treatment; none of the 3 dogs were clinically ill. Ten dogs (5 that received placebo and 5 that received caprofen) had negative pretreatment and positive posttreatment fecal occult blood test results.

Plasma and cerebrospinal fluid concentrations of 4-aminopyridine following intravenous injection and metered intrathecal delivery in canines.

Potassium channel blockade by 4-aminopyridine (4-AP) has been shown to initiate modest levels of functional recovery in spinal-injured dogs and people following intravenous administration; however, the relevant central nervous system (CNS) concentration mediating these effects is not known. We have determined the concentrations of 4-aminopyridine in plasma and cerebrospinal fluid following intravenous administration (0.5 mg/kg) in large (> 22 kg) dogs, using liquid column chromatography. Plasma levels are initially high (> 1 microgram/mL) and fall rapidly to levels less than 100 ng/mL by about 2 h postinjection. A characteristic secondary peak in plasma 4-AP is observed at about 1 h postinjection. Corresponding concentrations of 4-AP in CSF were relatively stable for nearly 2 h, never exceeding (as a mean) 50 ng/mL within the first 2 h postinjection. We suggest behavioral recovery in clinical cases of spinal cord injury in both dogs and humans is mediated by such low (< 50 ng/mL) concentrations of 4-AP bathing the lesion. Since the adverse side effects that accompany IV administration of the drug limit its potential clinical usefulness, we have evaluated the feasibility of an alternate route of administration, continuous metered delivery of 4-AP into the spinal cord's subarachnoid space. This is accomplished by using a surgically implantable pump and delivery catheter. The pump itself can be interrogated, and is fully programmable, by noninvasive telemetry. Intrathecal delivery rates of between 1 and 60 micrograms of 4-AP per hour never produced detectable levels of the drug in plasma or cervically sampled CSF in dogs independent of the amount or duration of infusion (hours to days). The levels of 4-AP in lumbar samples of CSF near the lumbar delivery site suggest a very steep gradient of the drug, with local concentrations easily reaching 1 microgram/mL or higher (10- to 20-fold higher than can be safely produced by IV administration). The most frequent adverse reaction to intrathecal 4-AP delivery was a mild hindlimb tremor, fully reversible following reduction in the rate of drug delivery or termination of delivery. This route of drug administration relative to clinical spinal cord injury is discussed.

A biomechanical comparison of techniques of flexor tendon repair.

Achieving satisfactory digital motion after suturing severed flexor tendons remains a challenge. Although a suture technique proposed by Savage is stronger in vitro than a true Kessler repair, the Savage technique has not been previously tested in vivo. We repaired 96 severed canine tendons using either of two modifications of the Kessler technique or the Savage technique. The tensile strength of these repairs were compared at 0, 1, 3, and 6 weeks after suturing. The Savage technique provided a significantly stronger repair than the "suture locking" method in vitro and at 1 and 3 weeks after repair. We found no significant differences between the tensile strengths of the Kessler-Tajima and suture-locking methods at any time.

Grafting in acute spinal cord injury: morphological and immunological aspects of transplanted adult rat enteric ganglia.

We have studied allogeneic transplants of adult rat enteric ganglia in order to evaluate their use as donor tissue for eventual autografts in rodent spinal cord injury models. Female Sprague-Dawley rats of similar weights served either as transplant donors or as recipients. A glass micropipette of 0.8 mm diameter was used to create a local penetrating injury of the lower thoracic spinal cord and the transplant material was pressure injected through the pipette within the neural parenchyma. Ganglia of the myenteric plexus adhering to the stratum longitudinal muscularis were dissected from portions of the jejunum and ileum. Following partial enzymatic digestion and mechanical disruption of the myenteric plexus and muscle tissue (labeled with adherent rhodamine conjugated microbeads), reaggregates of myenteric plexus and muscle were suspended in growth medium and cultured in vitro for one to two days prior to transplantation. Transplants were examined at three, four, six, and eight weeks after surgery. Some of the donor tissue was grown in vitro, in order to determine its cellular composition. These cultured explants were fixed after 10 days, and like myenteric plexus and muscle grafts, were stained histochemically for acetylcholinesterase and observed by fluorescence and light microscopy. At the earlier post-transplantation periods, grafts contained several clusters of enteric ganglion cells that were positive for acetylcholinesterase and exhibited ultrastructural features characteristic of the enteric nervous system. They had well-defined boundaries. Reactive astrocytes and their processes remained located within the host spinal cord adjacent to the boundary region of the grafts. Likewise, macrophages were located in areas abutting the graft. Newly formed vasculature penetrated the graft interior and appeared to be continuous with the host vessels. Grafts grown for at least eight weeks were characterized by interdigitating boundaries. Finger-like protrusions of graft tissue containing fibroblasts and collagen intermixed with adjacent gray and white matter of the host cord. Such transplants also had reactive astrocytes and ED1-positive macrophages. At this later stage, several groups of ganglion cells were identified that were intensely acetylcholinesterase-positive; however, only two of four grafts were recovered, whereas two of the transplants degenerated. We postulate that degeneration of allogeneic grafts may occur as a result of ongoing immune responses of the host which could be prevented by use of autogeneic enteric ganglia. Our studies show that fully differentiated enteric ganglia can survive transplantation to acutely injured spinal cord of adult rats.

Effects of applied electric fields on clinical cases of complete paraplegia in dogs.

A clinical trial of applied, slowly oscillating, weak electric fields was performed in dogs with naturally occurring spinal cord injuries due to intervertebral disc herniation. Criteria for admission to the study were: complete paraplegia, defined by neurological examination and electrophysiological testing; intact segmental reflexes; radiologic and myelographic evidence of spinal cord compression due to disc herniation and a focal lesion, without appreciable rostrocaudal spread of necrosis; weight less than 16 kg; onset of paralysis less than 1 month before surgery. The injured cord was exposed by laminectomy, and decompressed by aspiration of disc material. Active (n = 13) or sham (n = 11) stimulators were implanted subcutaneously, with platinum/iridium electrodes sutured to muscle several millimeters above the cord surface, at either end of the laminectomy site. Active stimulators delivered 200 µA of direct current, switching polarity every 15 min, for 3, 6, or 15 weeks. Neurological and electrophysiological examinations were repeated approximately 6 weeks and 6 months after implantation. Few complications were noted. None were attributable to current application. The group of dogs with active stimulators showed greater improvement in function than the group with sham implants, with a trend towards greater recovery in all neurological measures, and evoked potentials. The combined neurological score derived from these separate tests was significantly different from controls at 6 weeks and 6 months (P < 0.05). Application of slowly oscillating electric fields thus appears to be beneficial in the treatment of paraplegia in dogs.

Follow-up study of owner attitudes toward home care of paraplegic dogs.

A questionnaire was mailed to 30 owners of paraplegic dogs who had been caring for their dogs at home for 3 to 72 months. It was designed to collect information on demographic variables, duration of ownership and paralysis, age of the pet, pet/owner relationship, owner expectations and perceptions of the pet's quality of life, problems the pet experienced, effect that maintaining a paralyzed pet had on the owners' quality of life, and whether use of a cart was beneficial. Significant correlation was found between prior expectations that the pet would lead a high-quality life and perception that the pet, in fact, had a high quality of life during paralysis (r2 = 0.61, P = 0.01). Owners who had anticipated that extra work would be necessary to care for their paraplegic dog had a more positive attitude toward home care (r2 = 0.55, P = 0.03). Overall, owners involved in the study were satisfied with all aspects of maintaining paraplegic dogs at home. Our findings support the feasibility of dedicated owners successfully maintaining small (average body weight, 9 kg) paraplegic dogs at home for extended periods.

Arthrodesis for congenital flexural deformity of the metacarpophalangeal and metatarsophalangeal joints.

A llama, a miniature horse, and a miniature donkey with severe bilateral congenital flexural deformities of the metacarpophalangeal and metatarsophalangeal joints were treated successfully by arthrodesis with dynamic compression plating or external skeletal fixation. The flexor tendons were more taut than the suspensory ligaments and were transected. In the llama, the suspensory ligament was transected, and overcorrection caused ischemic necrosis of one distal limb and subluxation of the other fetlock joint. In the horse and donkey, the suspensory ligament was preserved and wedge osteotomy was performed to correct the deformity.

Transarticular application of external skeletal fixation.

External skeletal fixators provide a useful alternative to external coaptation techniques for immobilization of joints in selected patients. This is especially true when the orthopedic injury involves an open wound requiring daily treatment. Devices such as the K-E splint, the Rudy external fixator boot, and acrylic frame fixators are economical and effective for transarticular fixation in small animal patients. Clinical indications for these techniques have included fixation of tibial or radial fractures with a short distal fragment; arthrodesis of the elbow, carpus, stifle, or tarsus; protection of Achilles tendon repairs; protection of collateral ligament repairs of the tarsocrural joint; and protection when multiple ligaments of the stifle joint have been reconstructed. Use of contoured rods has facilitated transarticular application of type II Kirschner-Ehmer splints. Use of acrylic frames and the Rudy boot technique have extended safe use of transarticular external fixation to extremely small patients not accommodated by the Kirschner system alone. An understanding of the advantages and disadvantages inherent in each of these techniques is critical to proper selection of the best method for a given patient. Attention to the basic principles of pin selection, pin insertion, and frame design are needed for successful execution of the chosen technique.

Femoral head ostectomy in horses and cattle.

Femoral head ostectomy was performed in six horses, three ponies, and four cattle for treatment of fractures of the femoral capital physis, coxofemoral luxation, fractured acetabulum, or severe degenerative joint disease. The procedures were performed via a cranial approach that did not involve osteotomy of the greater trochanter. A dorsal approach for femoral head ostectomy via osteotomy of the greater trochanter was evaluated in three healthy adult ponies. Three animals (2 ponies, 1 calf) were euthanatized within a month and one horse was euthanatized at year 2 due to postoperative complications. Nine animals were discharged to owners and six of them fulfilled their intended functions of breeding, milking, and being kept as companions. One horse was lost to follow-up and two horses died of causes unrelated to the surgery. All surviving animals had a residual lameness that was described by owners as mild to moderate. None of the horses were used as riding animals. The mean age and weight of 10 animals that regained weight-bearing locomotion was 3.1 months and 84 kg; for three unsuccessful cases it was 34 months and 174 kg. We concluded that femoral head ostectomy was a viable salvage procedure for large animals with capital femoral physeal fracture, chronic coxofemoral luxation, or acetabular fracture. Surgical prognosis appeared to be favorable in young cattle and fair in young horses or ponies weighing less than 100 kg. Osteotomy of the greater trochanter resulted in superior exposure of the intact coxofemoral joint and allowed easier, less traumatic surgical luxation of the joint to facilitate femoral head ostectomy.

Effect of two muscle sling techniques on early morbidity after femoral head and neck excision in dogs.

Femoral head and neck excision was performed on the left coxofemoral joint of 18 dogs. The osteotomy site was padded with a partial-thickness biceps femoris muscle sling in one group of six dogs, and with a deep gluteal muscle flap in another group of six dogs. In a third group of six dogs, a partial-thickness biceps flap was created and sutured back in its original location. The dogs were monitored daily for 3 weeks for degree of lameness, range of motion, fever, and postoperative complications. Dogs with intra-articular muscle pads were less lame and had a greater range of motion in the treated hip than dogs without intra-articular muscle pads. Dogs with biceps slings showed more improvement in limb function and range of motion than did dogs with deep gluteal flaps. There were no significant differences in mean temperature elevations and no clinically significant postoperative complications. Use of a partial-thickness biceps muscle sling or a deep gluteal muscle flap decreased the chances of early postoperative morbidity and provided an earlier return to function. The partial-thickness biceps sling appeared to be more beneficial than the deep gluteal flap.

The effects of 4-aminopyridine on neurological deficits in chronic cases of traumatic spinal cord injury in dogs: a phase I clinical trial.

A Phase I trial of 4-aminopyridine (4-AP) was carried out in 39 dogs referred to the veterinary teaching hospital with naturally occurring traumatic paraplegia or paraparesis. The rationale for the study was provided by the observation that 4-AP restores conduction in demyelinated nerve fibers in experimental spinal cord injury. Most injuries (77%) resulted from degenerative disk disease, occurring at or near the thoracolumbar junction, and producing chronic, complete paraplegia. Neurological examination of each dog was recorded on videotape before and at intervals after administration of 4-AP. The drug was administered systemically in total doses between 0.5 and 1 mg/kg body weight. Three areas of neurological status changed significantly at 15-45 minutes following administration of 4-AP: (a) striking improvements in hindlimb placing occurred in 18 animals; (b) increased awareness of painful stimuli to the hindlimb in 10 animals; (c) partial recovery of the cutaneus trunci muscle reflex of the back skin in 9 animals. These effects reversed within a few hours of administration. Other animals (36%) showed no change in neurological signs except a slight enhancement of hindlimb reflex tone. Significant side effects were seen in 6 dogs receiving higher intravenous doses, with elevation of body temperature and apparent anxiety, leading to mild seizures in 3 of the animals. These seizures were controlled with diazepam. The results indicate that conduction block may contribute significantly to functional deficits in closed-cord injuries and that potassium channel blockade may prove to be a valid, if limited approach to therapeutic intervention in chronic paraplegia and paraparesis.

Colonic perforation in corticosteroid-treated dogs.

Clinical findings for 5 new cases of colonic perforation in corticosteroid-treated dogs were presented and 8 other cases from the literature were reviewed. Colonic perforation was a fatal complication in all 13 dogs, 12 of which had had recent major surgery. Ten dogs were neurosurgical patients, 1 dog received medical therapy for head trauma and nonambulatory paresis, and 2 dogs were operated on for non-neurologic conditions. Dexamethasone was the most frequently used corticosteroid, and 12 dogs received a mean cumulative dose of 6.4 mg/kg over an average period of 5.1 days. Depression, anorexia, and emesis, the most frequent signs attending colonic perforation, became evident 3 to 8 days after surgery. Signs preceded death by an average of 22.3 hours. Correct antemortem diagnosis (5 dogs) and surgical intervention (3 dogs) had no effect on eventual outcome (mortality = 100%). Colonic perforation most frequently developed in the proximal descending portion and always involved the antimesenteric border. Gross fecal contamination of the peritoneal cavity and acute generalized peritonitis were evident in all but one dog. Adhesions were observed at the lesion site in 6 dogs, but prevented gross soilage in only one dog.

Evaluation of autogeneic and allogeneic cortical chip grafting in a feline tibial nonunion model.

A feline tibial nonunion model was used to evaluate the efficacy of fresh autogeneic and deep-frozen allogeneic cortical chip grafts for bone repair. Rigidly fixated tibial ostectomies with a segmental defect length of 1 cm were grafted with 3 mm X 2 mm chips of cortical bone in 12 skeletally mature cats. Six cats were given fresh autogeneic bone (group A) and 6 were given deep-frozen allogeneic bone (group B). Bone healing was evaluated radiographically and histologically over a 12-week period. Consolidating bony callus and palpable stability of the tibia after bone plate removal were evident in all group A cats and 4 of the 6 group B cats by 12 weeks after surgery. Bone remodeling had progressed to intermediate restoration of diaphyseal structure in 2 cats of each group. Fibrous replacement of graft chips, absence of bony callus, and instability of the tibia after bone plate removal were evident in 1 group B cat at 12 weeks. The remaining group B cat was withdrawn from the study at 6 weeks due to loosening of the bone plate and screws.

Evaluation of Key's hypothesis in the feline tibia: an experimental model for augmented bone healing studies.

Key's hypothesis states that a segmental long bone defect 1.5 times the diaphyseal diameter exceeds the regenerative capacity of bone in skeletally mature dogs and results in nonunion. This hypothesis was evaluated in 5 adult cats with rigidly fixated segmental tibial ostectomies ranging from 1.25 to 1.52 times the diaphyseal diameter. Clinical, radiographic, and histologic data were obtained over a 12-week period. Healing was classified as mature bony union, clinical union, delayed union, or nonunion. Absence of a consolidating callus and instability after removal of fixation devices was found for all cats at 12 weeks. Scant formation of new bone within the gap was histologically evident for only 1 cat. In the remaining 4 cats, fibrous tissue and striated muscle predominated within the gap, and independent healing of the proximal and distal cut ends of the bone were observed. The occurrence of 4 nonunions and 1 equivocally delayed union indicated that Key's hypothesis overestimates the regenerative capacity of bone in the cat. Failure of this experimental model to produce clinical union within 12 weeks demonstrated that this model is a valid method of investigating augmented bone healing techniques that promote union within this period in the cat.